Temperature-based transient delivery of nucleic acids and proteins to cells and tissues

ABSTRACT

The present disclosure relates to methods for transiently activating temperature-sensitive agents in one or more cells, for example by contacting one or more cells with a temperature-sensitive agent and transiently incubating the cells at a permissive temperature for inducing an activity of the temperature-sensitive agent in the cells. Additionally, the present disclosure relates to methods of contacting one or more cells in a subject with a temperature-sensitive agent and then lowering the subject&#39;s core body temperature to a permissive temperature for inducing an activity of the temperature-sensitive agent in the cells. The disclosure also relates to methods of contacting one or more cells in a subject with a temperature-sensitive agent, maintaining the subject&#39;s surface body temperature at a permissive temperature for inducing an activity of the temperature-sensitive agent in the cells. Further disclosed are methods of treating a subject with a temperature-sensitive therapeutic agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2020/067506, filed Dec. 30, 2021, which claims the benefit of U.S.Provisional Application No. 62/992,700, filed Mar. 20, 2020, and U.S.Provisional Application No. 62/955,801, filed Dec. 31, 2019, thedisclosures of which are hereby incorporated by reference in theirentirety.

SUBMISSION OF SEQUENCE AS ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 699442001201SEQLIST.TXTdate recorded: Aug. 12, 2021, size: 25,620 bytes).

FIELD

The present disclosure relates to methods for transiently activating atemperature-sensitive agent (ts-agent) in one or more cells, for exampleby contacting one or more cells with a ts-agent and transientlyincubating the cells at a permissive temperature for inducing anactivity of the ts-agent in the cells. For ex vivo therapeuticstrategies, one or more cells are treated with a therapeutic ts-agent exvivo at the permissive temperature and the cells are subsequentlytransferred to a subject at the non-permissive temperature (e.g., thesubject's normal core body temperature). For in vivo therapeuticstrategies, a therapeutic ts-agent is delivered to a subject that ismaintained at the permissive temperature, permitting the therapeuticts-agent to function in vivo for a limited time before the ts-agent isturned off permanently when the subject's core body temperature returnsto normal, or when the subject's surface body temperature is raised(e.g., the non-permissive temperature). Alternatively, a therapeuticts-agent is delivered to a subject and the ts-agent is subsequentlytransiently activated by lowering the subject's core body temperature toa permissive temperature for inducing an activity of the therapeuticts-agent in cells of the subject.

BACKGROUND

Delivery of a therapeutic gene product to human cells, tissues, andorgans poses a great challenge. For traditional gene therapy, whichrequires the continuous expression of a gene to supplement the defect ofthe gene in a patient, this has been achieved by using a viral vectorsuch as a retrovirus, an adenovirus, or an adeno-associated virus.However, an equally important strategy gene therapy involves transient,short-term expression of a gene. For such applications, the persistentexpression of a gene is not required and may actually be deleterious tothe cells.

For example, CAS9 is a bacterial enzyme that cleaves DNA. It is animportant component of CRISPR/CAS9-based gene editing complex, which hasbeen considered for gene therapy. Both guide RNA and CAS9 can be encodedby genes on a single Sendai virus vector (Park et al., 2016). In orderto use the gene-editing system therapeutically, vectors containingCRISPR-CAS9 must be introduced into human cells or the human body.However, the continuous expression of CAS9 could cause the introductionof DNA breaks and mutations. Thus, it is desirable to have CAS9expressed for a short period of time, for example, on the order of hoursor a few days, rather than a week or more.

Another application for short-term expression of a gene is for cellularreprogramming. Recently, it has been shown that the ectopic expressionof a set of transcription factors can convert cells into therapeuticallyuseful cell types. For example, a set of three transcription factors canconvert pancreatic duct cells into insulin-secreting pancreaticbeta-cells (Zhou et al., 2008). Another set of transcription factors canconvert fibroblast cells into cardiomyocytes (Ieda et al., 2010). It isthought that in vivo delivery of these transcription factors into thehuman body could be used as one type of regenerative medicine. However,it is desirable to have these potent cell identity-changingtranscription factors expressed only transiently, as the continuousexpression of these potent transcription factors may cause harm.

As the above examples highlight, traditional gene therapy using viralvectors that lead to the continuous expression of a gene can beundesirable. For time-limited expression of a gene product, the deliveryof synthetic or in vitro-transcribed mRNA into cells has begun to beused (Warren et al., 2010). However, there are several problems withthese methodologies. For example, the amount of mRNA delivered to cells,tissues, and organs is limited, and thus, the amount of protein productmay not be sufficient for biologically meaningful effects in vivo.

Also, due to the fast turn-over of RNA, which normally lasts for only upto 12 hours (Warren et al., 2010; Goparaju et al., 2017), synthetic RNAmust be transfected into cells multiple times. For the forceddifferentiation of human pluripotent stem cells such as embryonic stemcells and induced pluripotent stem (iPS) cells, twice-dailytransfections over the course of several days are required (Akiyama etal., 2016; Goparaju et al. 2017). To generate iPS cells from humanfibroblast cells, daily transfection of a cocktail of synthetic RNAsmust continue for more than two weeks (Warren et al., 2010). This is notonly cumbersome, but also inefficient.

For the generation of iPS cells, this issue has been addressed by usingself-replicating RNA, which enables long-term expression after only onedelivery (Yoshioka et al., 2013). Self-replicating RNAs aresingle-stranded RNAs that are usually produced from alphaviruses (Joseet al., 2009), such as Venezuelan Equine Encephalitis Virus (VEEV),Sindbis Virus (SINV), and Semliki Forest Virus (SFV), by removing DNAencoding structural proteins that are required for virus particleformation (Petrakova et al., 2005). Self-replicating RNAs encodenonstructural proteins (nsPs), which function as an RNA-dependent RNApolymerase to replicate the self-replicating RNA itself and to produce atranscript for translation. Self-replicating RNAs can also include agene of interest (GOI) encoding a protein of interest, and other geneticelements. Due to its positive feedback production of RNAs,self-replicating RNAs can express the GOI at a high level.Self-replicating RNAs can be delivered to mammalian cells as a naked RNA(i.e., a synthetic RNA) or as a virus particle, which can be generatedby supplementing the missing virus structural proteins by packaginghelper cells.

The advantage of self-replicating RNA vectors are their self-replicatingfeature, which results in enhancement of expression levels of a GOI.However, one of the drawbacks of self-replicating RNA vectors to deliverRNA/protein to mammalian cells is their persistent expression. Usually,a positive feedback production of an RNA-dependent RNA polymerase and aGOI continues, which may result in the death of cells transfected with anaked RNA form of the self-replicating RNA or infected with a viral formof the self-replicating RNA.

Thus, what is needed in the art of gene therapy are tools for thetransient expression of a GOI encoding a protein of interest, such as atherapeutic agent or a foreign antigen (e.g., antigen of a pathogen). Inparticular, control of transcription and translation of RNA vectors andself-replicating RNA is desirable.

SUMMARY

Based on the necessity of having time-limited expression of a gene ofinterest (GOI), a transient gene product delivery system is required,where a nucleic acid or protein can be delivered to or expressed inspecific cells, in vitro or in vivo, where the amount of nucleicacid/protein is sufficient to have a biologically meaningful effect, andwhere transient expression can be turned off permanently after achievingthe biologically meaningful effect. In order to meet these and otherneeds, the present disclosure relates to methods for transientlyinducing an activity of a temperature-sensitive agent (ts-agent) such asa therapeutic ts-agent, either in a subject (in vivo) or in cells inculture (ex vivo). In some embodiments, the therapeutic ts-agent is usedin combination with mild therapeutic hypothermia. In other embodiments,the therapeutic ts-agent is used in combination with mild therapeutichyperthermia, or a localized application of heat. In some embodiments,the ts-agent is a ts-RNA molecule or ts-protein molecule. In someembodiments, the ts-agent is encoded by a heterologous nucleic acidinserted in a temperature-sensitive viral vector or a self-replicatingRNA. In some embodiments, the viral vector is selected from but notlimited to a Sendai virus vector, a retrovirus vector, an adeno virusvector, an adeno-associated virus vector, and an Alpha virus vector. Insome embodiments, the self-replicating RNA comprises an Alphavirusreplicon lacking a viral structural protein coding region. In someembodiments, the Alphavirus is selected from but not limited to aVenezuelan equine encephalitis virus, a Sindbis virus, and a SemlikiForrest virus. In some embodiments, the gene product of interest is notZSCAN4.

The foregoing and other objects and features of the disclosure willbecome more apparent from the following detailed description, whichproceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D depict the structure of the Venezuelan Equine EncephalitisVirus (VEEV) genome and locations of mutated regions. FIG. 1A shows aschematic representation of a wild type VEEV genome (TC-83 strain:complete genome 11,446 bp linear RNA: NCBI Accession: L01443.1 GI:323714). Genes of nonstructural proteins (nsP1, nsP2, nsP3, nsP4) encodeRNA-dependent RNA polymerase and genes of structural proteins encodeviral envelope proteins (C, E1, E2). 5′-UTR (5′-untranslated region) and3′-UTR (3′-untranslated region). The gene of the nsP2 protein, presentedas a bold box, was mutated to produce temperature sensitivity. FIG. 1Bshows a schematic representation of nsP2 with mutation 1(temperature-sensitive mutant 1: ts1). Five amino acids were insertedbetween amino acids 439 and 440. FIG. 1C shows a schematicrepresentation of nsP2 with mutation 2 (ts2). Five amino acids wereinserted between amino acids 586 and 587. FIG. 1D shows a schematicrepresentation of nsP2 with mutation 3 (ts3). Five amino acids wereinserted between amino acids 594 and 595.

FIGS. 2A-2C depict partial sequences of VEEV nsP2, corresponding to theregions mutated in ts1, ts2, and ts3. FIG. 2A shows the wild typesequence in comparison to mutant 1 (ts1), which includes a 15 nucleotideinsertion resulting in a 5 amino acid insertion. FIG. 2B shows the wildtype sequence in comparison to mutant 2 (ts2), which includes a 15nucleotide insertion resulting in a 5 amino acid insertion. FIG. 2Cshows the wild type sequence in comparison to mutant 3 (ts3), whichincludes a 15 nucleotide insertion resulting in a 5 amino acidinsertion.

FIG. 3 depicts partial nucleotide sequences for VEEV nsP1 of wild type(TC-83 strain) and mutant 4 (ts4), set forth as SEQ ID NO:19 and SEQ IDNO:20, respectively. The 5′-UTR and the 51-nt CSE (conserved sequenceelement) are shown in bold. Mutated nucleotides in ts4 are underlined.

FIGS. 4A and 4B depict testing temperature-sensitivity of srRNA1ts2 andsrRNA1ts3 at 30° C., 32° C., and 37° C. Wild type (srRNA1 wt-GFP) andmutant (srRNA1ts2-GFP, srRNA1ts3-GFP) self-replicating RNA (srRNA)vectors were generated. RNAs produced by in vitro transcription weretransfected into human induced pluripotent stem cells (ADSC-iPSC line).Cells were cultured in CO₂ incubators maintained at 30° C., 32° C., and37° C., respectively. Pictures of cells were obtained at 20 hours and 48hours, respectively. The upper panels show phase-contrast images and thelower panels show fluorescence images detecting expression of greenfluorescence protein (GFP). FIG. 4A shows results from transfection ofcells with srRNA1wt-GFP, srRNA1ts2-GFP, and srRNA1ts3-GFP RNA. FIG. 4Bshows results from transfection of cells with synthetic mRNA encodingGFP (synRNA-GFP).

FIG. 5 depicts testing temperature-sensitivity of srRNA1ts1 andsrRNA1ts2 at 32° C. Wild type (srRNA1 wt-GFP) and mutant (srRNA1ts2-GFPand srRNA1ts1-GFP) self-replicating RNA (srRNA) vectors were generated.RNAs produced by in vitro transcription were transfected into humaninduced pluripotent stem cells (ADSC-iPSC line). Cells were cultured inCO₂ incubators maintained at 32° C. Pictures of cells were obtained at24, 48, 72, 96, 120, 144, 168, 192, 240, 288 hours. For thesrRNA1ts1-GFP, only pictures of 24 hours and 168 hours were taken. Theupper panels show phase-contrast images and the lower panels showfluorescence images detecting expression of GFP.

FIG. 6 depicts testing temperature-sensitivity of srRNA1ts2 andsrRNA1ts4 at 32° C., 33° C., 37° C. Mutant (srRNA1ts2-GFP and srRNA1ts4-GFP) self-replicating RNA (srRNA) vectors were generated. RNAsproduced by in vitro transcription were transfected into human inducedpluripotent stem cells (ADSC-iPSC line). Cells were cultured in CO₂incubators maintained at 32° C., 33° C., 37° C., respectively. Picturesof cells were obtained at 20, 48, 96 hours. The upper panels showphase-contrast images and the lower panels show fluorescence imagesdetecting expression of GFP.

FIG. 7 depicts testing temperature-sensitivity of mutant srRNA1ts2-GFPmaintained at 32° C. RNAs produced by in vitro transcription of a mutantvector (srRNA1ts2-GFP) were transfected into human induced pluripotentstem cells (ADSC-iPSC line). Cells were cultured in CO₂ incubatorsmaintained at 32° C. The srRNA1ts2-GFP vector contains a puromycinN-acetyltransferase (pac) selection gene inserted after the “IRES”sequence, and thus, transfected cells can be selected using puromycin.The experiments were done in the absence (upper panel) or presence(lower panel) of 1 μg/ml of puromycin. Pictures of cells were obtainedat 24, 48, 96, 144, 168, 192 hours. The upper panels show phase-contrastimages and the lower panels show fluorescence images detectingexpression of GFP.

FIG. 8 depicts testing temperature-sensitivity of mutant srRNA1ts2-GFPwith a temperature switch from 32° C. to 37° C. at 24 hours. RNAsproduced by in vitro transcription of a mutant vector (srRNA1ts2-GFP)were transfected into human induced pluripotent stem cells (ADSC-iPSCline). Cells were cultured in CO₂ incubators maintained at 32° C. At 24hours, cells were transferred to a CO₂ incubator maintained at 37° C.The srRNA1ts2-GFP vector contains a puromycin N-acetyltransferase (pac)selection gene inserted after the “IRES” sequence, and thus, transfectedcells can be selected using puromycin. The experiments were done in theabsence (upper panel) or presence (lower panel) of 1 μg/ml of puromycin.Pictures of cells were obtained at 24, 48, 96, 144, 168, 192 hours. Theupper panels show phase-contrast images and the lower panels showfluorescence images detecting expression of GFP.

FIG. 9 depicts testing temperature-sensitivity of mutant srRNA1ts2-GFPwith a temperature switch from 32° C. to 37° C. at 48 hours. RNAsproduced by in vitro transcription of a mutant vector (srRNA1ts2-GFP)were transfected into human induced pluripotent stem cells (ADSC-iPSCline). Cells were cultured in CO₂ incubators maintained at 32° C. At 48hours, cells were transferred to CO₂ incubator maintained at 37° C. ThesrRNA1ts2-GFP vector contains a puromycin N-acetyltransferase (pac)selection gene inserted after the “IRES” sequence, and thus, transfectedcells can be selected using puromycin. The experiments were done in theabsence (upper panel) or presence (lower panel) of 1 μg/ml of puromycin.Pictures of cells were obtained at 24, 48, 96, 144, 168, 192 hours. Theupper panels show phase-contrast images and the lower panels showfluorescence images detecting expression of GFP.

FIG. 10 depicts testing temperature-sensitivity of mutant srRNA1ts2-GFPwith a temperature switch from 32° C. to 37° C. at 72 hours. RNAsproduced by in vitro transcription of a mutant vector (srRNA1ts2-GFP)were transfected into human induced pluripotent stem cells (ADSC-iPSCline). Cells were cultured in CO₂ incubators maintained at 32° C. At 72hours, cells were transferred to a CO₂ incubator maintained at 37° C.The srRNA1ts2-GFP vector contains a puromycin N-acetyltransferase (pac)selection gene inserted after the “IRES” sequence, and thus, transfectedcells can be selected using puromycin. The experiments were done in theabsence (upper panel) or presence (lower panel) of 1 μg/ml of puromycin.Pictures of cells were obtained at 24, 48, 96, 144, 168, 192 hours. Theupper panels show phase-contrast images and the lower panels showfluorescence images detecting expression of GFP.

FIGS. 11A-11D depict testing temperature-sensitivity of mutantsrRNA1ts2-GFP in fibroblast cells. RNAs produced by in vitrotranscription of a mutant vector (srRNA1ts2-GFP) were transfected intohuman newborn dermal fibroblast cells (HDFn line). Cells were culturedin CO₂ incubators maintained at 32° C. Pictures of cells were obtainedat 24, 48, and 96 hours. The upper panels show phase-contrast images andthe lower panels show fluorescence images detecting expression of GFP.FIGS. 11A and 11B depict transfections carried out using JetMessenger(Polyplus). Cells were cultured in standard media alone (FIG. 11A) orstandard media supplemented with 200 ng/ml of B18R (FIG. 11B). FIGS. 11Cand 11D depict transfections carried out using MessengerMax(ThermoFisher). Cells were cultured in standard media alone (FIG. 11C)or standard media supplemented with 200 ng/ml of B18R (FIG. 11D).

FIG. 12 depicts an alignment of amino acid sequences corresponding tonsP2 mutant 2 (ts2) of various alphavirus family members. The left paneldepicts an alignment (reproduced in part from FIG. 1 of Russo et al.,2006) of wild type sequences set forth as SEQ ID NOS:21-28, while theright panel depicts an alignment of mutant sequences set forth as SEQ IDNOS:29-36 including an insertion of 5 amino acids between the “β5” and“β6” (5^(th) and 6^(th) “β strands) in the secondary structure of nsP2.VEEV (Venezuelan equine encephalitis virus), Aura (Aura virus), WEEV(Western equine encephalitis virus), BFV (Barmah Forest virus), ONNV(O'nyong-nyong virus), RRV (Ross River virus), SFV (Semliki Forestvirus), and SINV (Sindbis virus).

FIG. 13 depicts a schematic diagram showing a typical ex vivo treatmentof cells with temperature-sensitive agents (ts-agents). Ts-agents suchas srRNAs or Sendai virus vectors, are functional at a permissivetemperature (e.g., 33° C.), but non-functional at a non-permissivetemperature (e.g., 37° C.). Target cells treated with the ts-agent arecultured at a permissive temperature for a certain duration (e.g., 3days), and then continue to be cultured at a non-permissive temperaturefor a certain duration (e.g., 10 days). Expected levels of RNA (orprotein translated from the RNA) of a gene of interest (GOI) increase ata permissive temperature and reach a high level. After switching to anon-permissive temperature, expected levels of RNA (or proteins)gradually decrease as transcription and translation cease.

FIG. 14 depicts a schematic diagram showing an exemplary ex vivotherapeutic procedure. Temperature-sensitive agents (ts-agents) such assrRNAs or Sendai virus vectors, are functional at a permissivetemperature (e.g., 33° C.), but non-functional at a non-permissivetemperature (e.g., 37° C.). Target cells are taken from a patient's body(autograft) and are incubated with the ts-agent ex vivo at a permissivetemperature, e.g., at 33° C., for a certain duration, e.g., 24 hours.Then, the target cells with ts-agents are transplanted in the patient.At a non-permissive temperature of 37° C., the ts-agent does notfunction inside the patient's body.

FIG. 15 depicts a schematic diagram showing another exemplary ex vivotherapeutic procedure. Temperature-sensitive agents (ts-agents) such assrRNAs or Sendai virus vectors, are functional at a permissivetemperature (e.g., 33° C.), but non-functional at a non-permissivetemperature (e.g., 37° C.). Target cells are taken from a donor's body(allograft) and are incubated with the ts-agent ex vivo at a permissivetemperature, e.g., at 33° C., for a certain duration, e.g., 24 hours.Then, the target cells with ts-agents are transplanted in a patient. Ata non-permissive temperature of 37° C., the ts-agent does not functioninside the patient's body.

FIG. 16 depicts a schematic diagram showing an exemplary semi in vivotherapeutic procedure. Temperature-sensitive agents (ts-agents) such assrRNAs or Sendai virus vectors, are functional at a permissivetemperature (e.g., 33° C.), but non-functional at a non-permissivetemperature (e.g., 37° C.). A patient undergoes a procedure fortherapeutic hypothermia and the patient's core body temperature ismaintained at a reduced temperature (e.g., 33° C.), which is lower thannormal body temperature (e.g., 37° C.). Target cells (either autologousor allogenic) are treated with the ts-agent ex vivo and immediatelyinfused into the patient's circulation or injected into an organ of thepatient. While the patient is maintained at the reduced temperature(e.g., 33° C.) for some time (e.g., 24 hours) the ts-agent isfunctional. Subsequently, the patient's core body temperature isreturned to normal temperature (37° C.), at which time the ts-agent isno longer functional.

FIG. 17 depicts a schematic diagram showing an exemplary in vivotherapeutic procedure. Temperature-sensitive agents (ts-agents) such assrRNAs or Sendai virus vectors, are functional at a permissivetemperature (e.g., 33° C.), but non-functional at a non-permissivetemperature (e.g., 37° C.). A patient undergoes a procedure fortherapeutic hypothermia and the patient's core body temperature ismaintained at a reduced temperature (e.g., 33° C.), which is lower thannormal body temperature (e.g., 37° C.). The ts-agent is directlydelivered systemically or to specific organs, tissues, or cell types.While the patient is maintained at the reduced temperature (e.g., 33°C.) for some time (e.g., 24 hours), the ts-agent is functional.Subsequently, the patient's core body temperature is returned to normaltemperature (37° C.), at which time the ts-agent is no longerfunctional.

FIG. 18 depicts differentiation of human ES/iPS cells into neurons. Aschematic representation of a typical experimental procedure is shown.Human ES/iPS cells were plated onto a cell culture dish on Day −1. OnDay 0, cells were transfected with srRNA1ts2-NGN3. Cells were culturedat 33° C. for 72 hours. On Day 3, cells were re-plated onto a newculture dish and then cell cultures were transferred to 37° C. Puromycinwas added to the medium on Day 3 for 24 hours. Phase contrastmicroscopic images were taken on Day 0 (before transfection), 1, 2, 3(before passaging), 4 (before medium change), 5, and 6. Magnified imageof Day 6 picture is also shown. Cells were fixed on Day 9 and used forimmunostaining with anti-TUBB3 (beta3-tubulins) (red signals), which isspecific to mature neurons (10× and 20× objective lens).

FIG. 19 depicts differentiation of human ES/iPS cells into vascularendothelial cells. A schematic representation of a typical experimentalprocedure is shown. Human ES/iPS cells were plated onto a cell culturedish on Day −1. On Day 0, cells were transfected with srRNA1ts2-ETV2.Cells were cultured at 33° C. for 72 hours. On Day 3, cell cultures weretransferred to 37° C. Puromycin was added to the medium on Day 3 for 48hours. (Lower Panel) Phase contrast microscopic images were taken on Day1, 2, 3, 4, 5, 6, 7, and 8. Cells were fixed on Day 8 and used forimmunostaining with anti-CD31 (10× and 20× objective lens).

FIG. 20 depicts a schematic diagram showing an exemplary in vivotherapeutic procedure. Temperature-sensitive agents (ts-agents) such assrRNAs or Sendai virus vectors, are functional at a permissivetemperature (e.g., 31-34° C.), but non-functional at a non-permissivetemperature (e.g., >37° C.). The temperature at or just below thesurface of a patient's body (surface body temperature), which is around31-34° C., is lower than the core body temperature of the patient, whichis around 37° C. The ts-agent is directly delivered by intradermal,subcutaneous, or intramuscular administration to a patient, where it isfunctional at the patient's surface body temperature. No further actionis required. Alternatively, when the function of the ts-agent is nolonger needed, the ts-agent can be rendered non-functional bytransiently increasing the patient's surface body temperature.

FIG. 21 depicts a schematic diagram showing exemplary srRNA1ts2 vectorsencoding the spike protein (or portions thereof) of severe acuterespiratory syndrome coronavirus-2 (SARS-CoV-2, also known as2019-nCoV). SARS-CoV-2 is the causative agent of coronavirus disease2019 (COVID-2019). Non-structural proteins (nsP1-nsP4) of the srRNA1ts2vector are required for replication and transcription of the RNA genome,while the gene of interest (GOI) encodes the spike protein or fragmentthereof. “srRNA1ts2-2019-nCoV-Spike” encodes the full-length spikeprotein (SEQ ID NO:41) of 2019-nCoV. “srRNA1ts2-2019-nCoV-RBD1” encodesa fusion protein (SEQ ID NO:42) including the signal peptide of CD5(residues 1-24) and the RBD of the spike protein of 2019-nCoV.“srRNA1ts2-2019-nCoV-RBD2” encodes a fusion protein (SEQ ID NO:43)including the signal peptide, RBD, transmembrane domain, and cytoplasmictail of the spike protein of 2019-nCoV. The amino acid sequence of theRBD of 2019-nCoV is set forth as SEQ ID NO:44. Abbreviations: SP (signalpeptide); RBD (receptor binding domain); TM (transmembrane domain); andCT (cytoplasmic tail).

FIG. 22 depicts a schematic diagram showing an exemplary in vivotherapeutic procedure. Temperature-sensitive agents (ts-agents) such assrRNAs or Sendai virus vectors, are functional at a permissivetemperature (e.g., 31-35° C.), but non-functional at a non-permissivetemperature (e.g., >37° C.). The temperature of airways of a patient'sbody (airway temperature), which is around 32° C. for nasal cavity andupper trachea, and 35° C. for subsegmental bronchi (McFadden et al.,1985), is lower than the core body temperature of the patient, which isaround 37° C. The ts-agent is directly delivered by nasal administration(e.g., insufflation, inhalation or instillation) to a patient, where itis functional at the patient's airway temperature. No further action isrequired. When the function of the ts-agent is no longer needed, thets-agent can be rendered non-functional by transiently increasing thepatient's airway temperature.

FIG. 23 depicts expression of a gene of interest in vivo as aconsequence of intradermal administration of RNA encoding the gene ofinterest (luciferase) to hind limbs of outbred mice.

FIGS. 24A-24B show the frequency of cytokine-secreting cells in samplesof splenocytes obtained from mice that had been immunized by intradermalinjection of a temperature-sensitive srRNA1ts2 RNA encoding a receptorbinding domain (RBD) of SARS-CoV-2 (srRNA1ts2-2019-CoV-RBD1) or aplacebo (buffer only). FIG. 24A shows the frequency of interferon-gamma(INF-7) spot-forming cells (SFC) and FIG. 24B shows the frequency ofinterleukin-4 (IL-4) SFC in splenocytes from immunized mice cultured inthe presence and absence of SARS-CoV-2 antigen as determined by ELISpotassays. Black bars (Stimulated) represent the splenocytes stimulated bya pool of 53 peptides (15mers with 11 amino acid overlaps) that coversSARS-CoV-2 RBD for 24 hours. Gray bars (Control) represent thesplenocytes without the stimulation. The average and standard deviation(error bars) of triplicate samples are shown.

FIG. 25A-25C show the levels of SARS-CoV-2 antigen-reactive serumimmunoglobulin G (IgG) of mice that had been immunized by intradermalinjection of a temperature-sensitive srRNA1ts2 RNA encoding a receptorbinding domain (RBD) of SARS-CoV-2 (srRNA1ts2-2019-CoV-RBD1) or aplacebo (buffer only). In brief, on Day 0 and Day 14, mice received aplacebo, or srRNA1ts2-2019-CoV-RBD1 in the presence and absence of anRNase inhibitor (black triangles). On Day 49, all mice received arecombinant RBD protein (open triangles). Asterisk (*) indicates IgGlevels higher than 3 (OD450). FIG. 25A shows results of mice thatreceived two doses of a placebo (buffer only). FIG. 25B shows results ofmice that received two doses of srRNA1ts2-2019-CoV-RBD1 RNA. FIG. 25Cshows results of mice that received two doses of srRNA1ts2-2019-CoV-RBD1RNA in combination with a RNase inhibitor. Asterisk (*) indicates IgGlevels higher than 3 (OD450). N=10 in all groups.

DETAILED DESCRIPTION

Overview

Applicant has demonstrated that cells can be cultured at a permissivetemperature for inducing an activity of a temperature-sensitivetherapeutic agent, and that the activity can lead to a therapeuticeffect in the cells. Moreover, the activity of the temperature-sensitivetherapeutic agent can be reduced or inhibited by subsequently incubatingthe cells at a non-permissive temperature. Applicant has also for thefirst time provided methods for use of temperature-sensitive agents(ts-agents) in vivo. The same types of ts-agents used in vitro can beused in vivo. For instance, after administration of a ts-agent to thecore of a subject, the subject's core body temperature can be lowered toa permissive temperature for inducing an activity of the ts-agent.Alternatively, after administration of a ts-agent to the surface(epidermis, dermis, hypodermis, or skeletal muscle) of a subject, thesubject's surface body temperature is maintained at a permissivetemperature for inducing an activity of the ts-agent. The subject'ssurface body temperature may be maintained naturally or artificially.These methods provide new ways to deliver and transiently activatetherapeutic agents such as nucleic acids and polypeptides. Inparticular, the present disclosure provides tools fortemperature-sensitive delivery of nucleic acids and proteins to cells,with the proviso that the nucleic acids and proteins are not ZSCAN4nucleic acids and proteins.

Accordingly, the present disclosure generally relates to methods oftransiently inducing an activity of a temperature-sensitive agent (e.g.,a temperature-sensitive therapeutic agent) in vitro. In someembodiments, one or more cells comprising a temperature-sensitivetherapeutic agent are cultured at a permissive temperature for inducingan activity of the temperature-sensitive therapeutic agent. The cellsare cultured at the permissive temperature for a period of timesufficient for the temperature-sensitive therapeutic agent to induce atherapeutic effect in the cells. The cells are then returned to anon-permissive temperature, wherein the non-permissive temperaturereduces or inhibits an activity of the temperature-sensitive therapeuticagent. In another embodiment, the one or more cells do not alreadycomprise a temperature-sensitive therapeutic agent, and are firstcontacted with a temperature-sensitive therapeutic agent. In someembodiments, after inducing a therapeutic effect in the one or morecells, the cells are administered to a subject in need thereof. In someembodiments, the one or more cells are isolated from a subject in needof treatment and after treating with a temperature-sensitive therapeuticagent, the cells are returned to said subject.

In another aspect, the present disclosure relates to methods oftransiently inducing an activity of a temperature-sensitive agent (e.g.,a temperature-sensitive therapeutic agent) in vivo. In some embodiments,one or more cells in a subject comprise a temperature-sensitivetherapeutic agent, and the subject's body temperature is lowered to apermissive temperature for a period of time sufficient for thetemperature-sensitive therapeutic agent to induce a therapeutic effectin the cells, and the subject's body temperature is then returned tonormal body temperature. In another embodiment, thetemperature-sensitive therapeutic agent is administered to the subject,either before or after the subject's body temperature is lowered to apermissive temperature.

Other aspects of the present disclosure relate to treating a disease orcondition by mobilizing bone marrow cells in a subject suffering from adisease or condition, the method comprises: isolating the mobilized bonemarrow cells from the subject, culturing the isolated bone marrow cellsat a temperature of about 33° C.±0.5° C., contacting said cells with atemperature-sensitive viral vector, such as Sendai viral vector, or atemperature-sensitive self-replicating RNA (srRNA), wherein the viralvector or the srRNA comprises a heterologous nucleic acid molecule,maintaining the contacted cells at about 33° C.±0.5° C. for a sufficientperiod of time, wherein the viral vector or the srRNA is capable ofreplicating at 33° C.±0.5° C. and replication of the viral vector or thesrRNA leads to increased expression of the heterologous nucleic acidmolecule, and engrafting the contacted cells into the subject to treatthe disease or condition. Alternatively, after isolating the mobilizedbone marrow cells from the subject, the isolated bone marrow cells arecontacted with a temperature-sensitive viral vector, such as Sendaiviral vector, or a temperature-sensitive srRNA before culturing thecells at a temperature of about 33° C.±0.5° C.

In another aspect, the present disclosure relates to treating a diseaseor condition by administering to a subject in need thereof atemperature-sensitive viral vector, such as Sendai viral vector, or atemperature-sensitive self-replicating RNA (srRNA), wherein the viralvector or the srRNA comprises a heterologous nucleic acid, lowering thesubject's core body temperature to about 33° C.±0.5° C., maintaining thesubject's core body temperature at about 33° C.±0.5° C. for a sufficientperiod of time, wherein the viral vector or the srRNA is capable ofreplicating at 33° C.±0.5° C. and replication of the viral vector or thesrRNA leads to increased expression of the heterologous nucleic acidmolecule, and allowing the subject's core body temperature to return tonormal. Alternatively, lowering the subject's core body temperature toabout 33° C.±0.5° C. is done prior administering a temperature-sensitiveviral vector, such as Sendai viral vector, or a temperature-sensitivesrRNA.

References and claims to methods for treating a disease or condition byadministering a ts-agent or cells comprising the ts-agent to a subject,in their general and specific forms likewise related to:

a) the use of a ts-agent or cells comprising the ts-agent for themanufacture of a medicament for the treatment of a disease or condition;and

b) pharmaceutical compositions comprising a ts-agent or cells comprisingthe ts-agent for the treatment of a disease or condition.

In some embodiments of the methods of the proceeding paragraphs, theheterologous nucleic acid comprises a gene of interest (GOI) or encodesa protein of interest. In preferred embodiments, the protein of interestis a therapeutic agent. In some embodiments, the GOI is adominant-negative form of the GOI, or an artificial gene encoding anartificial protein (e.g., hybrid protein made by fusing differentprotein domains). In some embodiments, the heterologous nucleic acidcomprises a non-coding RNA, an siRNA, or an shRNA. In other embodiments,the heterologous nucleic acid comprises an endonuclease editing system.In some embodiments, the endonuclease editing system is selected frombut not limited to a ZFN system, a TALENs system, and a CRISPR/CAS9system. In some embodiments, the protein of interest is selected frombut not limited to human neurogenin-3 (NGN3), human ETS translocationvariant 2 (ETV2), brain-derived neurotrophic factor (BDNF) and nervegrowth factor (NGF). In some embodiments, the protein of interest iserythropoietin (EPO) or granulocyte colony stimulating factor (G-CSF).In other embodiments, the protein of interest is an enzyme, such asadenosine deaminase (ADA), for enzyme replacement therapy. In someembodiments, the protein of interest is an antigen encoded by apathogenic organism, such as a virus, a protozoan, or a bacteria, forthe purpose of vaccination against an infectious disease.

Definitions

As used herein and in the appended claims, the singular forms “a,” “an”and “the” include plural forms unless otherwise indicated. For example,“a polynucleotide” includes one or more polynucleotides.

The phrase “comprising” as used herein is open-ended, indicating thatsuch embodiments may include additional elements. In contrast, thephrase “consisting of” is closed, indicating that such embodiments donot include additional elements (except for trace impurities). Thephrase “consisting essentially of” is partially closed, indicating thatsuch embodiments may further comprise elements that do not materiallychange the basic characteristics of such embodiments. It is understoodthat aspects and embodiments described herein as “comprising” include“consisting of” and “consisting essentially of” embodiments.

The term “about” as used herein in reference to a value other thantemperature, encompasses from 90% to 110% of that value (e.g., about 30minutes refers to 27 min to 33 min), unless otherwise indicated. Whenuse in reference to temperature in Celsius, about encompasses −1° C. to+1° C. of that value (e.g., about 37° C. refers to 36° C. to 38° C.),unless otherwise indicated. In contrast, the use of plus or minuswithout more, delineates the indicated range (e.g., 33° C.±0.5° C.refers to 32.5° C. to 33.5° C.).

As used herein, numerical ranges are inclusive of the numbers definedthe range (e.g., 12-18 nucleotides encompasses 12, 13, 14, 15, 16, 17and 18 nucleotides).

The terms “isolated” and “purified” as used herein refers to an object(e.g., a cell) that is removed (e.g., separated) from its environment(e.g., cell culture, biological sample, etc.). “Isolated” objects are atleast 50% free, preferably 75% free, more preferably at least 90% free,and most preferably at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%) freefrom other components with which they are associated.

The terms “individual” and “subject” refer to mammals. “Mammals”include, but are not limited to, humans, non-human primates (e.g.,monkeys), farm animals, sport animals, rodents (e.g., mice and rats) andpets (e.g., dogs and cats).

The term “dose” as used herein in reference to a pharmaceuticalcomposition refers to a measured portion of the composition taken by(administered to or received by) a subject at any one time.

The term “treating” a disease or a condition refer to executing aprotocol, which may include administering one or more pharmaceuticalcompositions to an individual (human or other mammal), in an effort toalleviate signs or symptoms of the disease. Thus, “treating” or“treatment” does not require complete alleviation of signs or symptoms,does not require a cure, and specifically includes protocols that haveonly a palliative effect on the individual. As used herein, and aswell-understood in the art, “treatment” is an approach for obtainingbeneficial or desired results, including clinical results. Beneficial ordesired clinical results include, but are not limited to, alleviation oramelioration of one or more symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, preventing spread ofdisease, delay or slowing of disease progression, amelioration orpalliation of the disease state, and remission (whether partial ortotal).

“Stimulating” an immune response, means increasing the immune response,which can arise from eliciting a de novo immune response (e.g., as aconsequence of an initial vaccination regimen) or enhancing an existingimmune response (e.g., as a consequence of a booster vaccinationregimen). In some embodiments, stimulating an immune response includesbut is not limited to one or more of the group consisting of:stimulating CD4+T helper cell proliferation; stimulating cytokineproduction; stimulating B lymphocyte proliferation; stimulating antibodyproduction; stimulating CD8+ cytotoxic T cell proliferation; andstimulating cytolysis of infected cells. In some preferred embodiments,stimulating an immune response comprises increasing an antigen-specificantibody response in the subject. Preferably, increasing theantigen-specific antibody response comprises increasing theconcentration of antigen-specific antibodies at least 2, 3 or 4 foldhigher than a pre-administration level. In some embodiments, increasingthe antigen-specific antibody response comprises increasing theconcentration of antigen-specific antibodies above a minimum level,preferably above a seroprotective level.

Temperature-Sensitive Agents

Certain aspects of the present disclosure relate to transiently inducingan activity of a temperature-sensitive agent (e.g., atemperature-sensitive therapeutic agent) in one or more cells. Anactivity of the temperature-sensitive agent refers to any desiredactivation, replication, or increased expression of the agent. As usedherein, the term “temperature-sensitive agent” refers to any nucleicacid or polypeptide that has different levels of functionality atdifferent temperatures. Exemplary temperature-sensitive agents include,without limitation, temperature-sensitive viral vectors,temperature-sensitive self-replicating RNAs, and temperature-sensitivepolypeptides.

As used herein, the term “permissive temperature” refers to anytemperature at which the activity of a temperature-sensitive agent ofthe present disclosure is induced. Typically, a permissive temperatureis not the normal body temperature of a subject. The normal bodytemperature of a human subject is about 37° C.±0.5° C. Depending on thetemperature-sensitive agent, a permissive temperature may be atemperature that is higher or lower than the normal body temperature ofa subject. In some aspects, the permissive temperature for thetemperature-sensitive agent ranges from 30° C. to 36° C. In someembodiments, the permissive temperature is from about 31PC to about 35°C., or 32° C. to 34° C. (33° C.±1.0° C.). In some preferred embodiments,the permissive temperature is 33° C.±0.5° C. It follows that in someembodiments, the non-permissive temperature for thetemperature-sensitive self-replicating RNAs of the present disclosure isabove 36° C. In some preferred embodiments, the non-permissivetemperature is 37° C.±0.5° C.

In some embodiments, the activity of the temperature-sensitive agentinduced at a permissive temperature is reduced or inhibited at anon-permissive temperature. The term “non-permissive temperature”, asused herein, refers to any temperature at which an activity of atemperature-sensitive agent of the present disclosure is not induced. Atemperature-sensitive agent is not induced when an activity of thetemperature-sensitive agent is at least 95% less, at least 90% less, atleast 85% less, at least 80% less, at least 75% less, or at least 50%less than the level of activity at the optimal permissive temperature.Typically, a non-permissive temperature is the normal body temperatureof a subject. Depending on the temperature-sensitive agent, anon-permissive temperature may also be a temperature that is higher orlower than the normal body temperature of a subject.

Temperature-Sensitive Viral Vectors

In certain embodiments, a temperature-sensitive therapeutic agent of thepresent disclosure may comprise a temperature-sensitive viral vector. Insome embodiments, an activity of the temperature-sensitive viral vectorinduced at a permissive temperature may include replication of thevector. As used herein, the term “temperature-sensitive viral vector”refers to any viral vector that has different levels of functionality atdifferent temperatures. Exemplary temperature-sensitive viral vectorsinclude, without limitation, Sendai virus vectors, Adeno associatedvirus vectors, retrovirus vectors, or alphavirus vectors. Exemplarytemperature-sensitive alphavirus vectors include, without limitation,Venezuelan Equine Encephalitis virus vectors, Sindbis virus vectors, andSemliki Forrest virus vectors.

In some embodiments of the present disclosure, a temperature-sensitiveviral vector comprises a heterologous nucleic acid (e.g., foreignnucleic acid in relation to the viral vector). A nucleic acid maycomprise a genetic element. As used herein, the term “genetic element”refers to any nucleic acid that encodes an RNA or polypeptide ofinterest. Exemplary genetic elements include, without limitation, a geneof interest (GOI), a dominant-negative form of a gene of interest, anartificial gene that encodes an artificial protein such as a hybridprotein made by fusing different protein domains, a non-coding RNA, ansiRNA, an shRNA, and an endonuclease editing system. In someembodiments, the endonuclease editing system is selected from a ZFNsystem, a TALENs system, and a CRISPR/CAS9 system. In some embodiments,the GOI encodes a protein selected from but not limited to humanneurogenin-3 (NGN3), human ETS translocation variant 2 (ETV2),brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF).In some embodiments, the protein of interest is erythropoietin (EPO) orgranulocyte colony stimulating factor (G-CSF). In other embodiments, theprotein of interest is an enzyme, such as adenosine deaminase (ADA), forenzyme replacement therapy. In some embodiments, the protein of interestis an antigen encoded by a pathogenic organism, such as a virus, aprotozoan, or a bacteria, for the purpose of vaccination against aninfectious disease.

The permissive temperature for the temperature-sensitive viral vectorsof the present disclosure typically ranges from 30° C. to 36° C. or from38° C. to 50° C. In some embodiments, the permissive temperature is fromabout 31° C. to about 35° C., or 32° C. to 34° C. (33° C.±1.0° C.). Insome preferred embodiments, the permissive temperature is 33° C.±0.5° C.It follows that in some embodiments, the non-permissive temperature forthe temperature-sensitive viral vectors of the present disclosure isabove 36° C. and below 38° C. In some preferred embodiments, thenon-permissive temperature is 37° C.±0.5° C.

As disclosed herein, cells may be maintained at a permissive temperaturefor a period of time sufficient for the temperature-sensitive agent toinduce an effect. In some embodiments, a temperature-sensitive viralvector comprises a genetic element, and an effect includes increasedexpression of the genetic element, wherein expression of the geneticelement results in production of an RNA or polypeptide that creates abiological effect in the cells. In some preferred embodiments, theeffect is a therapeutic effect.

Temperature-Sensitive Self-Replicating RNAs

In certain embodiments, a temperature-sensitive therapeutic agent of thepresent disclosure may comprise a temperature-sensitive self-replicatingRNA. As used herein, the term “temperature-sensitive self-replicatingRNA” refers to any self-replicating RNA that has different levels offunctionality at different temperatures.

In some embodiments, temperature-sensitive self-replicating RNAs arecreated by engineering self-replicating RNAs, which are single-strandedRNAs that are usually made from the Alphavirus such as Venezuelan EquineEncephalitis Virus (VEEV), Sindbis Virus (SINV), and Semliki ForestVirus (SFV), by removing DNAs encoding structural proteins that arerequired for virus particle formation (Petrakova et al., 2005). In someembodiments, self-replicating RNAs encode nonstructural proteins (nsPs),which function as an RNA-dependent RNA polymerase to replicate theself-replicating RNA itself and to produce a transcript for translation.In some embodiments, self-replicating RNAs can also comprise a gene ofinterest (GOI) encoding a protein of interest, and other geneticelements. Without wishing to be bound by theory, in some embodiments,due to its positive feedback production of RNAs, self-replicating RNAscan express the GOI at a high level. In some embodiments, atemperature-sensitive self-replicating RNA may be created by mutatinggenes encoding nsPs.

In some embodiments, temperature-sensitive self-replicating RNAs can bedelivered to mammalian cells as a naked RNA (i.e., a synthetic RNA). Insome embodiments, temperature-sensitive self-replicating RNAs can bedelivered to mammalian cells as a naked RNA (i.e., a synthetic RNA)encapsulated by nanoparticles. In some embodiments, nanoparticles areengineered to target specific cell types, tissues, organs, cancers,tumors, or abnormal cells. In some embodiments, temperature-sensitiveself-replicating RNAs can be delivered to mammalian cells as a virusparticle, which can be generated by supplementing the missing virusstructural proteins by packaging helper cells. In some embodiments,virus particles are engineered to target specific cell types, tissues,organs, cancers, tumors, or abnormal cells.

When the temperature-sensitive agent is a temperature-sensitiveself-replicating RNA, an activity of the temperature-sensitiveself-replicating RNA induced at a permissive temperature may includereplication of the RNA.

In some aspects, the permissive temperature for temperature-sensitiveself-replicating RNAs of the present disclosure typically ranges from30° C. to 36° C. In some embodiments, the permissive temperature is fromabout 31° C. to about 35° C., or 32° C. to 34° C. (33° C.±1.0° C.). Insome preferred embodiments, the permissive temperature is 33° C.±0.5° C.It follows that in some embodiments, the non-permissive temperature forthe temperature-sensitive self-replicating RNAs of the presentdisclosure is above 36° C. In some preferred embodiments, thenon-permissive temperature is 37° C.±0.5° C.

In other aspects, the permissive temperature for temperature-sensitiveself-replicating RNAs of the present disclosure typically ranges from38° C. to 50° C. It follows that in some embodiments, the non-permissivetemperature for the temperature-sensitive self-replicating RNAs of thepresent disclosure is above 36° C. and below 38° C. In some preferredembodiments, the non-permissive temperature is 37° C.±0.5° C.

Temperature-Sensitive Polypeptides

In certain embodiments, a temperature-sensitive therapeutic agent of thepresent disclosure may comprise a temperature-sensitive polypeptide. Asused herein, the term “temperature-sensitive polypeptide” refers to anytemperature-sensitive polypeptide that has different levels offunctionality at different temperatures. In some embodiments, thetemperature-sensitive polypeptide may be a temperature-sensitiveantibody. In other embodiments, the temperature-sensitive polypeptide isselected from but not limited to a transcription factor, a growthfactors, a cell surface marker, a cell fusion protein, an epigeneticmodifier, an enzyme, and a structural protein.

When the temperature-sensitive agent is a temperature-sensitivepolypeptide, an activity of the temperature-sensitive protein induced ata permissive temperature may include a conformational change (e.g.,change to the structure or shape) of the protein.

The permissive temperature for the temperature-sensitive polypeptides ofthe present disclosure typically ranges from 30° C. to 36° C. or from38° C. to 50° C. In some embodiments, the permissive temperature is fromabout 31° C. to about 35° C., or from 32° C. to 34° C. (33° C.±1.0° C.).In some preferred embodiments, the permissive temperature is 33° C.±0.5°C. It follows that in some embodiments, the non-permissive temperaturefor the temperature-sensitive self-replicating polypeptides of thepresent disclosure is above 36° C. and below 38° C. In some preferredembodiments, the non-permissive temperature is 37° C.±0.5° C.

Various aspects of the present disclosure relate to substantiallypurified polypeptides. A substantially purified polypeptide may refer toa polypeptide which is substantially free of other polypeptides, lipids,carbohydrates or other materials with which it is naturally associated.In one embodiment, the polypeptide is at least 50%, for example at least80% free of other polypeptides, lipids, carbohydrates or other materialswith which it is naturally associated. In another embodiment, thepolypeptide is at least 90% free of other polypeptides, lipids,carbohydrates or other materials with which it is naturally associated.In yet another embodiment, the polypeptide is at least 95% free of otherpolypeptides, lipids, carbohydrates or other materials with which it isnaturally associated.

Nucleic Acids and Polypeptides

Certain aspects of the present disclosure relate to transiently inducingan activity of a temperature-sensitive therapeutic agent in one or morecells, wherein the activity leads to increased expression of a nucleicacid molecule. In some embodiments, the nucleic acid is apolynucleotide. A polynucleotide may refer to a nucleic acid sequence(such as a linear sequence) of any length. Therefore, a polynucleotideincludes oligonucleotides, and also gene sequences found in chromosomes.An oligonucleotide is a plurality of joined nucleotides joined by nativephosphodiester bonds. An oligonucleotide is a polynucleotide of between6 and 300 nucleotides in length. An oligonucleotide analog refers tomoieties that function similarly to oligonucleotides but havenon-naturally occurring portions. For example, oligonucleotide analogscan contain non-naturally occurring portions, such as altered sugarmoieties or inter-sugar linkages, such as a phosphorothioateoligodeoxynucleotide. Functional analogs of naturally occurringpolynucleotides can bind to RNA or DNA, and include peptide nucleic acid(PNA) molecules.

In certain embodiments, the nucleic acid molecules or polynucleotidesencode a genetic element. These polynucleotides include DNA, cDNA andRNA sequences, such as mRNA sequences, which encode a gene of interest.A coding sequence may be operably linked to a heterologous promoter todirect transcription of the genetic element. A promoter may refer tonucleic acid control sequences which direct transcription of a nucleicacid. A promoter includes necessary nucleic acid sequences near thestart site of transcription. A promoter also optionally includes distalenhancer or repressor elements. A constitutive promoter is a promoterthat is continuously active and is not subject to regulation by externalsignals or molecules. In contrast, the activity of an inducible promoteris regulated by an external signal or molecule (for example, atranscription factor). A first nucleic acid sequence is operably linkedto a second nucleic acid sequence when the first nucleic acid sequenceis placed in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked nucleic acid sequences arecontiguous and where necessary to join two protein coding regions, inthe same reading frame. A heterologous polypeptide or polynucleotiderefers to a polypeptide or polynucleotide derived from a differentsource or species. A promoter includes necessary nucleic acid sequencesnear the start site of transcription, such as, in the case of apolymerase II type promoter, a TATA element. A promoter also optionallyincludes distal enhancer or repressor elements which can be located asmuch as several thousand base pairs from the start site oftranscription. In one example, the promoter is a constitutive promoter,such as the CAG-promoter (Niwa et al., Gene 108(2):193-9, 1991), or thephosphoglycerate kinase (PGK)-promoter. In some embodiments, thepromoter is an inducible promoter such as a tetracycline-induciblepromoter (Masui et al., Nucleic Acids Res. 33:e43, 2005). Otherexemplary promoters that can be used to drive expression of a geneticelement include but are not limited to: lac system, the trp system, thetac system, the trc system, major operator and promoter regions of phagelambda, the control region of fd coat protein, the early and latepromoters of SV40, promoters derived from polyoma, adenovirus,retrovirus, baculovirus and simian virus, the promoter for3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, andthe promoter of the yeast alpha-mating factors. Genetic elements of thepresent disclosure may be under the control of a constitutive promoter,an inducible promoter, or any other suitable promoter described hereinor other suitable promoter that will be readily recognized by oneskilled in the art.

In some embodiments, inducing an activity of a temperature-sensitiveagent leads to increased expression of a nucleic acid or a polypeptide,which may include increased expression by at least 1.5 fold, at least1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, atleast 2.0 fold, at least 2.1 fold, at least 2.1 fold, at least 2.2 fold,at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least3.0 fold, at least 3.5 fold, at least 4.0 fold, at least 4.5 fold, atleast 5.0 fold, at least 5.5 fold, at least 6.0 fold, at least 6.5 fold,at least 7.0 fold, at least 7.5 fold, at least 8.0 fold, at least 8.5fold, at least 9.0 fold, at least 9.5 fold, at least 10 fold, at least50 fold, at least 100 fold, at least 200 fold, at least 300 fold, atleast 400 fold, at least 500 fold, at least 600 fold, at least 700 fold,at least 800 fold, at least 900 fold, at least 1,000 fold, at least2,000 fold, at least 3,000 fold, at least 4,000 fold, at least 5,000fold, at least 6,000 fold, at least 7,000 fold, at least 8,000 fold, atleast 9,000 fold, at least 10,000 fold, at least 25,000 fold, at least50,000 fold, at least 75,000 fold, at least 100,000 fold, at least125,000 fold, at least 150,000 fold, at least 175,000 fold, at least200,000 fold, at least 225,000 fold, at least 250,000 fold, at least275,000 fold, at least 300,000 fold, at least 325,000 fold, at least350,000 fold, at least 375,000 fold, at least 400,000 fold, at least425,000 fold, at least 450,000 fold, at least 475,000 fold, at least500,000 fold, at least 750,000 fold, or at least 1,000,000 fold, forexample, relative to the polynucleotide or polypeptide expression in ahuman cell that has not been contacted with the agent.

Various aspects of the present disclosure relate to isolated entities,such as isolated nucleic acids or synthetic mRNA molecules. An isolatednucleic acid has been substantially separated or purified away fromother nucleic acid sequences and from the cell of the organism in whichthe nucleic acid naturally occurs, i.e., other chromosomal andextrachromosomal DNA and RNA. The term “isolated” thus encompassesnucleic acids purified by standard nucleic acid purification methods.The term also embraces nucleic acids prepared by recombinant expressionin a host cell as well as chemically synthesized nucleic acids.Similarly, isolated polypeptides have been substantially separated orpurified from other polypeptides of the cells of an organism in whichthe protein naturally occurs, and encompasses polypeptides prepared byrecombination expression in a host cell as well as chemicallysynthesized polypeptides. Similarly, isolated cells have beensubstantially separated away from other cell types.

Methods of Introducing Temperature-Sensitive Agents into Cells

In some embodiments the one or more cells are contacted with atemperature-sensitive agent. Contacting may refer to placement in directphysical association, including both in solid and liquid form.“Contacting” may be used interchangeably with “exposed.” In some cases,“contacting” includes transfecting, such as transfecting a nucleic acidmolecule into a cell. In some cases “contacting” includes introducingthe temperature-sensitive agent into one or more cells.

In some embodiments, temperature-sensitive agents are polynucleotides(e.g. self-replicating RNAs), and polynucleotides are introduced intocells. Introducing a nucleic acid molecule or a protein into a cellencompasses any means of delivering the nucleic acid molecule or proteininto the cell. For example, nucleic acid molecules can be transfected,transduced or electroporated into a cell. In some embodimentstemperature-sensitive agents are polypeptides (e.g.temperature-sensitive polypeptides), and polypeptides are introducedinto cells. Delivery of polypeptides into cells can be achieved, forexample, by fusing the protein to a cell-penetrating peptide, such as apeptide with a protein transduction domain (e.g., HIV-1 Tat), or apoly-arginine peptide tag (Fuchs and Raines, Protein Science14:1538-1544, 2005). Protein transduction domains may refer to smallcationic peptides that facilitate entry of larger molecules (proteins,nucleic acid molecules etc.) into a cell by a mechanism that isindependent of classical endocytosis. A poly-arginine peptide tag mayrefer to a short peptide (generally 7 to 11 residues) comprised ofarginine residues that facilitates delivery of larger molecules (such asproteins and nucleic acid molecules) into cells (see, for example, Fuchsand Raines, Protein Science 14:1538-1544, 2005).

Introduction of nucleic acids into cells with a temperature-sensitiveagent may involve using a temperature-sensitive viral vector (such asintegrating or non-integrating viral vectors) or a temperature-sensitiveplasmid vector. Each of these methods has been described in the art andis therefore within the capabilities of one of skill in the art. A briefsummary of each method that can be used to nucleic acid to a human cellis provided herein. A vector may refer to a nucleic acid molecule asintroduced into a host cell, thereby producing a transformed host cell.A vector may include nucleic acid sequences that permit it to replicatein a host cell, such as an origin of replication (DNA sequences thatparticipate in initiating DNA synthesis). For example, an expressionvector contains the necessary regulatory sequences to allowtranscription and translation of inserted gene or genes. A vector mayalso include one or more selectable marker genes and other geneticelements known in the art. Vectors may include, for example, virusvectors and plasmid vectors.

Permissive Temperatures

Incubating One or More Cells at a Permissive Temperature

Certain aspects of the present disclosure relate to transiently inducingan activity of a temperature-sensitive agent in one or more cells byincubating the cells at a permissive temperature for inducing anactivity of the temperature-sensitive agent. In some embodiments, thepermissive temperature may be higher or lower than the standard cellculture temperature. For example, human and rodent cells are typicallycultured at a temperature of about 37° C. Accordingly, in someembodiments the permissive temperature may be lower than about 36.5° C.For example, in some embodiments the cells are cultured at a permissivetemperature of 36° C., 35.5° C., 35° C., 34.5° C., 34° C., 33.5° C., 33°C., 32.5° C., 32° C., 31.5° C., 31° C., 30.5° C., or 30° C. In somepreferred embodiments, the permissive temperature is from 30° C. to 36°C., or from 31° C. to 35° C., or from 32° C. to 34° C., or from 32.5° C.to 33.5° C. In some embodiments, the permissive temperature is greaterthan or equal to (lower limit) 30° C., 31° C., 32° C., 33° C., 34° C.,or 35° C., and is less than or equal to (upper limit) 36° C., 35° C.,34° C., 33° C., 32° C. or 31° C.

In other embodiments, the permissive temperature maybe higher than about37.5° C. For example, in some embodiments the cells are cultured at apermissive temperature of 38° C., 38.5° C., 39° C., 39.5° C., 40° C.,40.5° C., 41° C., 41.5° C., 42° C., 42.5° C., 43° C., 43.5° C., 44° C.,44.5° C., 45° C., 45.5° C., 46° C., 46.5° C., 47° C., 47.5° C., 48° C.,48.5° C., 49° C., 49.5° C., or 50° C.

In some embodiments, after incubating at a permissive temperature, theone or more cells are cultured at a non-permissive temperature whereinthe activity of the temperature-sensitive agent is reduced or inhibited.For example, replication of temperature-sensitive viral vectors can beinhibited, replication of temperature-sensitive self-replicating RNAscan be inhibited, and conformational changes to temperature-sensitivepolypeptides can be inhibited. This temperature shifting allows theactivity of the temperature-sensitive agent to be transiently inducedand then inhibited. In other embodiments, the one or more cells areadministered to a subject after being cultured at a permissivetemperature. The one or more cells may be administered to a subjectdirectly from culture at a permissive temperature, or may first betransferred from a permissive temperature to a non-permissivetemperature in culture and then administered to a subject. In certainembodiments, the temperature-sensitive agent is subsequently degraded.For example, non-integrating temperature-sensitive viral vectors, RNAs,and polypeptides will be degraded.

Lowering the Core Body Temperature of a Subject to a PermissiveTemperature

Certain aspects of the present disclosure relate to transiently inducingan activity of a temperature-sensitive therapeutic agent in cells in asubject by lowering the subject's core body temperature to a permissivetemperature for inducing the activity of the temperature-sensitiveagent. In some embodiments the subject's core body temperature islowered using a target-temperature management (TTM) procedure. A TTMprocedure is designed to achieve and maintain a specific bodytemperature in a subject for a duration of time. Such procedures havepreviously been used therapeutically to reduce the negative effectsresulting from various acute health issues such as heart attacks andstrokes. Equipment and general methods of using them are known in theart and can be used in the methods described herein. The procedure canbe carried out using a number of methods, including cooling catheters,cooling blankets, and application of ice around the body.

After lowering the subject's core body temperature to a permissivetemperature, the subject's core body temperature is maintained at thepermissive temperature for a time sufficient to induce an activity ofthe temperature-sensitive agent. The subject's core body temperature issubsequently returned to normal core body temperature, which is anon-permissive temperature wherein the activity of thetemperature-sensitive agent is reduced or inhibited. In certainembodiments the temperature-sensitive agent is subsequently degraded.For example, non-integrating temperature-sensitive viral vectors, RNAs,and polypeptides will be degraded at the non-permissive temperature. Asused herein, the term “body temperature” refers to “core bodytemperature”, unless context clearly indicates otherwise.

Maintaining the Surface Body Temperature of a Subject at a PermissiveTemperature

Certain aspects of the present disclosure relate to exploiting thenatural temperature differences in regions of a subject's body. Forexample, the temperature at or near the surface of a human subject'sbody (surface body temperature) is around 31-34° C., which is lower thanthe core body temperature of the human subject, which is around 37° C.As used herein, the “surface” of a subject's body refers to one or moreof the epidermis, dermis, hypodermis, or muscle. The “skin” of asubject's body refers to one or both of the epidermis and dermis. Thus,suitable routes of administration to the epidermis, dermis, orhypodermis of a subject's body include intradermal and subcutaneousadministration. A suitable route of administration to muscle near thesurface of a subject's body is intramuscular administration.

For instance, the ts-agent is directly delivered to a specific area ofthe skin of a subject (in the case of vaccination) or to a broader areaof the skin of a subject (in the case of treatment of a skin disease).The skin temperature (about 31-34° C.) is a permissive temperature forthe ts-agent, permitting the ts-agent to function. No further action isrequired for the long-term expression of GOI. If termination of thefunction of the ts-agent is need or desired, the temperature of thetreated skin is increased and transiently maintained at non-permissivetemperature (>37° C.) by local application of heat (e.g., heat patch orheating blanket) or by mild therapeutic hyperthermia (e.g., warm bath orhot sauna). This therapeutic procedure is safe in that the ts-agentfunctions only in the intended area of the body, because the core bodytemperature is a non-permissive temperature (about 37° C.). In someembodiments, should the surface body temperature of the subject behigher than normal, the surface body temperature is lowered to match thepermissive temperature of the ts-agent.

Maintaining the Upper Respiratory Tract Temperature of a Subject at aPermissive Temperature

Like the surface body temperature of a human subject, the temperature ofthe upper respiratory tract and upper trachea of a human subject is apermissive temperature for the ts-agent, permitting the ts-agent tofunction. That is, the temperature of the nasal cavity and upper tracheaof a human subject is about 32° C., and the temperature of thesubsegmental bronchi of a human subject is about 35° C. (McFadden etal., 1985). As such, ts-agents administered intranasally to cells of theupper respiratory tract (nasal cavity, pharnyx, and/or larnyx) and/orupper trachea of a human patient are functional without lowering thecore body temperature of the human patient. Intranasal administrationmay be done by insufflation, inhalation or instillation.

Non-Permissive Temperatures

Incubating One or More Cells at a Non-Permissive Temperature

In vitro culture of cells is usually carried out at the normal bodytemperature of the subject from which the cells are derived. Forexample, mammalian cells, such as human cells and mouse cells, areusually cultured at about 37° C. Certain aspects of the presentdisclosure relate to a temperature-sensitive agent that does notfunction (e.g., does not replicate or express genes) at the normal bodytemperature of the subject. Thus, the normal body temperature of thesubject is a non-permissive temperature for the temperature-sensitiveagent. In some preferred embodiments, the non-permissive temperature is37° C.±0.5° C.

Normal Core Body Temperature of a Subject

In some embodiments, a temperature-sensitive agent, cells contacted witha temperature-sensitive agent, or cells carrying a temperature-sensitiveagent, are introduced into a subject body that is maintained at thenormal body temperature. Certain aspects of the present disclosurerelate to a temperature-sensitive agent that does not function, e.g.,replicate or express genes, at this normal body temperature(non-permissive temperature) of the organism. This feature provides asafety mechanism that prevent the undesirable action or reactivation ofthe temperature-sensitive agent during the life-course of the subject.

Human Cells

Certain aspects of the present disclosure relate to transiently inducingan activity of a temperature-sensitive therapeutic agent in one or morehuman cells, including without limitation, human adult cells. In certainembodiments, the one or more human cells are in a subject in need oftreatment with the therapeutic agent.

Various human cells find use in the methods described herein. Asdisclosed herein, the term “human cell(s)” refers to any cell(s) foundthroughout the human body during and after embryonic development, suchas human embryonic cells, stem cells, pluripotent cells, differentiatedcells, mature cells, somatic cells, and adult cells. In someembodiments, human cells of the present disclosure are human adultcells. As disclosed herein, the term “human adult cell(s)” refers to anycell(s) found throughout the human body after embryonic development(i.e., non-embryonic cells). Human cells of the present disclosureinclude, without limitation, sperm cells, oocyte cells, fertilizedoocytes (i.e., zygotes), embryonic cells, mature cells, differentiatedcells, somatic cells, progenitor cells, embryonic stem (ES) cells,induced pluripotent stem (iPS) cells, adult stem cells, somatic stemcells, and tissue stem cells. Adult stem cells, which are also known assomatic stem cells or tissue stem cells, may refer to undifferentiatedcells, found throughout the body after embryonic development, whichmultiply by cell division to replenish dying cells and regeneratedamaged tissues. Progenitor cells may refer to oligopotent or unipotentcells that differentiate into a specific type of cell or cell lineage.Progenitor cells are similar to stem cells but are more differentiatedand exhibit limited self-renewal. Exemplary adult stem cells, tissuestem cells, and/or progenitor cells may include, without limitation,hematopoietic stem cells, mesenchymal stem cells, adipose stem cells,neuronal stem cells, intestinal stem cells, skin stem cells, and germcells (such as, sperm cells and oocytes).

Human cells may also include, without limitation, somatic cells, maturecells, and differentiated cells. Somatic cells may refer to any cell ofthe body, including, without limitation, germ cells, tissue stem cells,progenitor cells, induced pluripotent stem (iPS) cells, anddifferentiated cells. Exemplary somatic cells, mature cells, and/ordifferentiated cells may include, without limitation, epidermal cells,fibroblasts, lymphocytes, hepatocytes, epithelial cells, myocytes,chondrocytes, osteocytes, adipocytes, cardiomyocytes, pancreatic pcells, keratinocytes, erythrocytes, peripheral blood cells, bone marrowcells, neurocytes, astrocytes, and germ cells. Germ cells may refer tothe cells that give rise to the gametes (i.e., eggs and sperm) oforganisms that reproduce sexually. In certain embodiments, germ cellsinclude, without limitation, oocytes, and sperm cells. In someembodiment, somatic cells, mature cells, and/or differentiated cells ofthe present disclosure also include, without limitation, preimplantationembryos.

Human cells may also include, without limitation, cells derived fromcord blood, hematopoietic stem cells, CD34+ cells, mesenchymal stemcells, vascular endothelial stem cells, tissue stem cells, granulocytes,lymphocytes, T-cells, B-cells, monocytes, macrophages, dendritic cells,red blood cells, reticulocytes, and megakaryocytes. Human cells may alsoinclude, without limitation, abnormal cells of human origins, such ascancer cells, tumor cells, malignant cells, benign cells, hyperplasticcells, hypoplastic cells, and atypical cells. Human cells may alsoinclude, without limitation, diploid cells, haploid cells, tetraploidcells, polyploid cells, cells with karyotype abnormalities, cells withchromosome abnormalities, cells with mutated genes, cells with abnormaltelomere lengths, cells with short telomeres, and cells with longtelomeres. Human cells may also include, without limitation, cells withepigenetic abnormalities, such as cells with hypomethylated genomicregions, cells with hypermethylated genomics regions, cells with theabnormal histone modifications such as acetylation and methylation.

In some embodiments, the subjects of the present disclosure arenon-human animals. Non-human animals may refer to all animals other thanhumans. A non-human animal includes, but is not limited to, a non-humanprimate, a farm animal such as swine, cattle, and poultry, a sportanimal or pet such as dogs, cats, horses, hamsters, rodents, such asmice, or a zoo animal such as lions, tigers or bears. In one embodiment,the non-human animal is a mouse.

Therapeutic Uses of Temperature-Sensitive Agents

Temperature-sensitive agents of the present disclosure may beadministered by any suitable method known in the art, including, withoutlimitation, by oral administration, sublingual administration, buccaladministration, topical administration, rectal administration, viainhalation, transdermal administration, subcutaneous injection,intravenous (IV) injection, intra-arterial injection, intramuscularinjection, intracardiac injection, intraosseous injection, intradermalinjection, intraperitoneal injection, transmucosal administration,vaginal administration, intravitreal administration, intra-articularadministration, peri-articular administration, local administration,epicutaneous administration, or any combinations thereof. In someembodiments, the composition is administered by subcutaneous injectionand/or intravenous injection. In some embodiments, the composition isadministered by injection into the spleen of the subject.

In some aspects, the methods of the present disclosure involve the useof a therapeutically effective amount of a temperature-sensitive agent.A therapeutically effective amount of an agent may refer to the amountof a therapeutic agent sufficient to achieve the intended purpose. Forexample, a therapeutically effective amount of a temperature-sensitiveagent in a human cell to treat a disease or condition is an amountsufficient to reduce the disease or condition, or one or more symptomsof the disease or condition. A therapeutically effective amount may insome examples not treat the disease or condition, or symptoms of thedisease or condition 100%. However, a decrease in any known feature orsymptom of the disease or condition, such as a decrease of at least 25%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95% can be therapeutic.

The therapeutically effective amount of a given therapeutic agent willvary with factors such as the nature of the agent, the route ofadministration, the size and/or age of the subject to receive thetherapeutic agent, and the purpose of the administration. Thetherapeutically effective amount in each individual case can bedetermined empirically without undue experimentation by a skilledartisan according to established methods in the art.

A subject may refer to living multi-cellular vertebrate organisms, acategory that includes human and non-human mammals. In some embodiments,the subject is a human. Subjects that can be treated using the methodsprovided herein may include mammalian subjects, such as a veterinary orhuman subject. Subjects may include fertilized eggs, zygotes,preimplantation embryos, embryos, fetus, newborns, infants, children,and/or adults. In some embodiments, the subject to be treated isselected, such as selecting a subject that would benefit from a therapy,particularly therapy that includes administration of atemperature-sensitive agent of the present disclosure.

Pharmaceutical compositions of the present disclosure comprise ats-agent, such as a therapeutic ts-agent, and one or more additionalcompounds. As used herein, the terms “pharmaceutically acceptablecarrier” and “pharmaceutically acceptable vehicle” refer to the one ormore additional compound(s) (i.e., compounds other than the ts-agent).Pharmaceutically acceptable carriers suitable for use in the presentdisclosure are conventional. In particular, compositions andformulations suitable for pharmaceutical delivery of compositionscomprising a temperature-sensitive agent are as previously described(see, e.g., Gennaro, A. R. (editor) Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 18th edition (1990); and Felton, L. A.(editor) Remington Essentials of Pharmaceutics, Pharmaceutical Press,London, United Kingdom, 1′ edition, (2013)).

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example, sodiumacetate or sorbitan monolaurate. In some embodiments, pharmaceuticalcompositions of the present disclosure comprise a ts-agent, such as atherapeutic ts-agent, and one or more additional compounds, whichfacilitate the incorporation of ts-agent into cells. In the case ofRNA-based ts-agent, ts-agent is encapsulated in nanoparticles. In someinstances, nanoparticles is lipid-based (e.g., lipofectamine).

The therapeutic dose and regimen most appropriate for patient treatmentwill vary with diseases or conditions to be treated, and according tothe patient's weight and other parameters. An effective dosage andtreatment protocol can be determined by conventional means, startingwith a low dose in laboratory animals and then increasing the dosagewhile monitoring the effects, and systematically varying the dosageregimen. Numerous factors can be taken into consideration by a clinicianwhen determining an optimal dosage for a given subject. Factors includethe size of the patient, the age of the patient, the general conditionof the patient, the particular disease being treated, the severity ofthe disease, the presence of other drugs in the patient, and the like.The trial dosages would be chosen after consideration of the results ofanimal studies and the clinical literature.

Mobilizing Bone Marrow Cells

In some embodiments, the methods include mobilizing bone marrow cells(including, without limitation, CD34+ cells, hematopoietic stem cells,mesenchymal stem cells, endothelial stem cells) to the spleen andperipheral blood of the subject. In some embodiments, the methodsinclude administering a therapeutically effective amount of atemperature-sensitive agent (e.g., a temperature-sensitive therapeuticagent) of the present disclosure under conditions suitable for thetemperature-sensitive agent to deliver a nucleic acid to one or morebone marrow cells (including, without limitation, CD34+ cells,hematopoietic stem cells, mesenchymal stem cells, endothelial stemcells) in the spleen.

In some embodiments of the methods disclosed herein, mobilizing bonemarrow cells (including, without limitation, CD34+ cells, hematopoieticstem cells, mesenchymal stem cells, endothelial stem cells) to thespleen and peripheral blood comprises administering to the subject atherapeutically effective amount of a cytokine and/or achemotherapeutic. In some embodiments, mobilizing bone marrow cells(including, without limitation, CD34+ cells, hematopoietic stem cells,mesenchymal stem cells, endothelial stem cells) to the spleen andperipheral blood comprises administering to the subject atherapeutically effective amount of a cytokine. In some embodiments,mobilizing bone marrow cells (including, without limitation, CD34+cells, hematopoietic stem cells, mesenchymal stem cells, endothelialstem cells) to the spleen and peripheral blood comprises administeringto the subject a therapeutically effective amount of a chemotherapeutic.In some embodiments, mobilizing bone marrow cells (including, withoutlimitation, CD34+ cells, hematopoietic stem cells, mesenchymal stemcells, endothelial stem cells) to the spleen and peripheral bloodcomprises administering to the subject a therapeutically effectiveamount of a cytokine and a chemotherapeutic. Cytokines and/orchemotherapeutics may be administered by any suitable method known inthe art, including, without limitation, by oral administration,sublingual administration, buccal administration, topicaladministration, rectal administration, via inhalation, transdermaladministration, subcutaneous injection, intravenous (IV) injection,intra-arterial injection, intramuscular injection, intracardiacinjection, intraosseous injection, intradermal injection,intraperitoneal injection, transmucosal administration, vaginaladministration, intravitreal administration, intra-articularadministration, peri-articular administration, local administration,epicutaneous administration, or any combinations thereof. In someembodiments, the cytokine and/or chemokine is administered bysubcutaneous injection and/or intravenous injection.

In some embodiments, the bone marrow cells (including, withoutlimitation, CD34+ cells, hematopoietic stem cells, mesenchymal stemcells, endothelial stem cells) of the subject are mobilized at least 4weeks before, at least 3 weeks before, at least 2 weeks before, at least1 week before, at least 6 days before, at least 5 days before, at least4 days before, at least 3 days before, at least 2 days before, at least1 day before, less than 1 day before, at least 18 hours before, at least16 hours before, at least 12 hours before, at least 8 hours before, atleast 6 hour before, or at least 1 hour before administration of thecomposition (e.g., any nanoparticle composition as described herein). Insome embodiments, the bone marrow cells (including, without limitation,CD34+ cells, hematopoietic stem cells, mesenchymal stem cells,endothelial stem cells) of the subject are mobilized for sevenconsecutive days, five consecutive days, four consecutive days, threeconsecutive days, two consecutive days, or for one day beforeadministration of the composition. In some embodiments, the bone marrowcells (including, without limitation, CD34+ cells, hematopoietic stemcells, mesenchymal stem cells, endothelial stem cells) of the subjectare mobilized concurrently with administration of the composition.

Any cytokine capable of mobilizing bone marrow cells (including, withoutlimitation, CD34+ cells, hematopoietic stem cells, mesenchymal stemcells, endothelial stem cells) known in the art may be used, including,without limitation, granulocyte-colony stimulating factor (G-CSF),granulocyte-macrophage colony stimulating factor (GM-CSF),erythropoietin (EPO), thrombopoietin (TPO), stem cell factor (SCF),parathyroid hormone (PTH), and any combinations thereof. In someembodiments, the cytokine is G-CSF.

In some embodiments, the G-CSF is administered to the subject at aconcentration of about 0.1 μg/kg to about 100 μg/kg, or about 1.0 μg/kgto about 10 μg/kg. In some embodiments, the G-CSF is administered to thesubject at a concentration of about 2.5 μg/kg. In some embodiments, theG-CSF is administered to the subject at a concentration of about 10μg/kg.

Any chemotherapeutic capable of mobilizing bone marrow cells (including,without limitation, CD34+ cells, hematopoietic stem cells, mesenchymalstem cells, endothelial stem cells) known in the art may be used,including, without limitation, plerixafor, cyclophosphamide (CY),paclitaxel, etoposide, POL6326, BKT-140, TG-0054, NOX-A12, SEW2871, BIO5192, bortezomib, SB-251353, FG-4497, and any combinations thereof. Insome embodiments, the chemotherapeutic is plerixafor.

In some embodiments, the plerixafor is administered to the subject at aconcentration of about 1 μg/kg to about 1000 μg/kg, or about 75 μg/kg toabout 500 μg/kg. In some embodiments, the plerixafor is administered tothe subject at a concentration of about 150 μg/kg. In some embodiments,the plerixafor is administered to the subject at a concentration ofabout 240 μg/kg.

In some embodiments, mobilizing bone marrow cells (including, withoutlimitation, CD34+ cells, hematopoietic stem cells, mesenchymal stemcells, endothelial stem cells) to the spleen and peripheral bloodcomprises administering a therapeutically effective amount of G-CSF anda therapeutically effective amount of plerixafor. In some embodiments,the G-CSF and plerixafor are co-administered to the subject. In someembodiments, the G-CSF and plerixafor are co-administered to the subjectfor one day, two days, three days, four days, or more. In someembodiments, the G-CSF is administered to the subject prior to theplerixafor. In some embodiments, the G-CSF is administered to thesubject one day, two days, three days, four days or more prior to theplerixafor. In some embodiments, the G-CSF is administered to thesubject one day, two days, three days, four days or more prior to theplerixafor, and G-CSF and plerixafor are then co-administered to thesubject for one day, two days, three days, four days or more. In someembodiments, the plerixafor is administered to the subject prior to theG-CSF. In some embodiments, the plerixafor is administered to thesubject one day, two days, three days, four days or more prior to theG-CSF. In some embodiments, the plerixafor is administered to thesubject one day, two days, three days, four days or more prior to theG-CSF, and G-CSF and plerixafor are then co-administered to the subjectfor one day, two days, three days, four days or more.

In some embodiments, one or more human cells are contacted with atemperature-sensitive agent (e.g., a temperature-sensitive therapeuticagent) that delivers a nucleic acid to the one or more human cells. Insome embodiments, the nucleic acid comprises a gene of interest orencodes a protein of interest.

In some aspects, the methods of the present disclosure involve the useof a therapeutically amount of a temperature-sensitive agent (e.g., atemperature-sensitive therapeutic agent) that delivers a nucleic acid tocells of a subject in vitro or in vivo. A therapeutically effectiveamount of an agent may refer to the amount of a therapeutic agentsufficient to achieve the intended purpose. For example, atherapeutically effective amount of a temperature-sensitive agent (e.g.,a temperature-sensitive therapeutic agent) that delivers a nucleic acidto a human cell to treat a disease or condition is an amount sufficientto reduce the disease or condition, or one or more symptoms of thedisease or condition. A therapeutically effective amount may in someexamples not treat the disease or condition, or symptoms of the diseaseor condition 100%. However, a decrease in any known feature or symptomof the disease or condition, such as a decrease of at least 25%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, or atleast 95% can be therapeutic.

In another example, a therapeutically effective amount of a cytokineand/or chemokine capable of mobilizing bone marrow cells (including,without limitation, CD34+ cells, hematopoietic stem cells, mesenchymalstem cells, endothelial stem cells) in a subject is an amount sufficientto induce mobilization of one or more bone marrow cells (including,without limitation, CD34+ cells, hematopoietic stem cells, mesenchymalstem cells, endothelial stem cells) from the bone marrow into theperipheral blood.

The therapeutically effective amount of a given temperature-sensitiveagent (e.g., a temperature-sensitive therapeutic agent) will vary withfactors such as the nature of the agent, the route of administration,the size and/or age of the subject to receive the therapeutic agent, andthe purpose of the administration. The therapeutically effective amountin each individual case can be determined empirically without undueexperimentation by a skilled artisan according to established methods inthe art.

A subject may refer to living multi-cellular vertebrate organisms, acategory that includes human and non-human mammals. In some embodiments,the subject is a human. Subjects that can be treated using the methodsprovided herein may include mammalian subjects, such as a veterinary orhuman subject. Subjects may include a fetus, newborns, infants,children, and/or adults. In some embodiments, the subject to be treatedis selected, such as selecting a subject that would benefit from atherapy.

Treating Diseases and Disorders

Examples of disorders or diseases that can benefit from administrationof a temperature-sensitive agent (e.g., a temperature-sensitivetherapeutic agent) include those disorders or diseases that areassociated with gene mutation(s), abnormal telomere length, or abnormalepigenetic modification(s). Further examples of disorders or diseasesthat can benefit from administration of a temperature-sensitive agent(e.g., a temperature-sensitive therapeutic agent) include cancer,autoimmune diseases, and diseases in which cell regeneration isbeneficial, such as neurologic injuries or a neurodegenerativedisorders, as well as blindness and deafness.

Cancers include malignant tumors that are characterized by abnormal oruncontrolled cell growth. Cancers are frequently associated with genemutations and aberrant telomere regulation. Exemplary cancers that canbenefit from treatment with a ts-agent include but are not limited tocancers of the heart (e.g., sarcoma (angiosarcoma, fibrosarcoma,rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma andteratoma), lung (e.g., bronchogenic carcinoma (squamous cell,undifferentiated small cell, undifferentiated large cell,adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma,sarcoma, lymphoma, chondromatous hamartoma, mesothelioma);gastrointestinal tract (e.g., esophagus (squamous cell carcinoma,adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma), genitourinary tract (e.g., kidney (adenocarcinoma, Wilms'tumor, nephroblastoma, lymphoma, leukemia), bladder and urethra(squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma),prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma,embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma,interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors,lipoma), liver (e.g., hepatoma (hepatocellular carcinoma),cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellularadenoma, hemangioma), bone (e.g., osteogenic sarcoma (osteosarcoma),fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing'ssarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma,malignant giant cell tumor, chordoma, osteochondroma (osteocartilaginousexostoses), benign chondroma, chondroblastoma, chondromyxofibroma,osteoid osteoma and giant cell tumors), nervous system (e.g., skull(osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges(meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma,medulloblastoma, glioma, ependymoma, germinoma, pinealoma, glioblastomamultiforme, oligodendroglioma, schwannoma, retinoblastoma, congenitaltumors), spinal cord (neurofibroma, meningioma, glioma, sarcoma),gynecological cancers (e.g., uterus (endometrial carcinoma), cervix(cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovariancarcinoma, serous cystadenocarcinoma, mucinous cystadenocarcinoma,endometrioid tumors, Brenner tumor, clear cell carcinoma, unclassifiedcarcinoma, granulosa-thecal cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma,embryonal rhabdomyosarcoma, fallopian tubes (carcinoma)), hematologiccancers (e.g., blood (myeloid leukemia (acute and chronic), acutelymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferativediseases, multiple myeloma, myelodysplastic syndrome), Hodgkin'sdisease, non-Hodgkin's lymphoma (malignant lymphoma)), skin (e.g.,malignant melanoma, basal cell carcinoma, squamous cell carcinoma,Kaposi's sarcoma, moles, dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis), and adrenal glands (e.g.,neuroblastoma).

An autoimmune diseases result from an aberrant immune response, such asthe production of antibodies or cytotoxic T cells specific for aself-antigen or a subject's own cells or tissues. In some instances, theautoimmune disease is restricted to certain organs (e.g., inthyroiditis) or can involve a particular tissue in different places(e.g., Goodpasture's disease). Exemplary autoimmune diseases that canbenefit from treatment with a ts-agent include but are not limited torheumatoid arthritis, juvenile oligoarthritis, collagen-inducedarthritis, adjuvant-induced arthritis, Sjogren's syndrome, multiplesclerosis, experimental autoimmune encephalomyelitis, inflammatory boweldisease (for example, Crohn's disease, ulcerative colitis), autoimmunegastric atrophy, pemphigus vulgaris, psoriasis, vitiligo, type 1diabetes, non-obese diabetes, myasthenia gravis, Grave's disease,Hashimoto's thyroiditis, sclerosing cholangitis, sclerosingsialadenitis, systemic lupus erythematosis, autoimmune thrombocytopeniapurpura, Goodpasture's syndrome, Addison's disease, systemic sclerosis,polymyositis, dermatomyositis, autoimmune hemolytic anemia, andpernicious anemia.

In some embodiments, the subject is one who has suffered a neurologicinjury or suffers from a neurodegenerative disorder. A neurologicalinjury may refer to a trauma to the nervous system (such as to the brainor spinal cord or particular neurons), which adversely affects themovement and/or memory of the injured patient. For example, suchpatients may suffer from dysarthria (a motor speech disorder),hemiparesis or hemiplegia. Neurologic injuries can result from a traumato the nervous system (such as to the brain or spinal cord or particularneurons), which adversely affects the movement and/or memory of theinjured patient. Such traumas may be caused by an infectious agent(e.g., a bacterium or virus), a toxin, an injury due to a fall or othertype of accident, or genetic disorder, or for other unknown reasons.Accordingly, in some embodiments, a temperature-sensitive agent (e.g., atemperature-sensitive therapeutic agent) of the present disclosure thattemperature-sensitive may be used to treat a neurologic injury in asubject, by modulating tissue stem cells in the nervous system of apatient that has suffered a neurologic injury, where modulating tissuestem cells in the nervous system produces neurons and glial cells,thereby repairing defects in nervous system. In some embodiments, atemperature-sensitive agent encoding various neurotrophic factors suchas brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF),neurotrophin-3, neurotrophin-4, ciliary neurotrophic factor, glial cellline-derived neurotrophic factor (GDNF) may be used to treat suchpatients. In some embodiments, the patient may have suffered aneurologic injury, such as a brain or spinal cord injury resulting froman accident, or from a stroke.

A neurodegenerative disease is a condition in which cells of the brainand/or spinal cord are lost. Neurodegenerative diseases result fromdeterioration of neurons or their myelin sheath which over time leads todysfunction and disabilities. Conditions that result can cause problemswith movement (such as ataxia) and with memory (such as dementia).Accordingly, in some embodiments, a temperature-sensitive agent (e.g., atemperature-sensitive therapeutic agent) of the present disclosure maybe used to treat a neurodegenerative disease in a subject, by modulatingtissue stem cells in the nervous system of a patient suffering from aneurodegenerative disease, where modulating tissue stem cells in thenervous system produces neurons and glial cells, thereby repairingdefects in nervous system. In some embodiments, the agent modulates thenervous system of the subject and revert the degenerative conditions ofthe disease. Exemplary neurodegenerative diseases include but are notlimited to: adrenoleukodystrophy (ALD), alcoholism, Alexander's disease,Alper's disease, Alzheimer's disease, amyotrophic lateral sclerosis (LouGehrig's Disease), ataxia telangiectasia, Batten disease (also known asSpielmeyer-Vogt-Sjogren-Batten disease), bovine spongiformencephalopathy (BSE), Canavan disease, cerebral palsy, Cockaynesyndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, familialfatal insomnia, frontotemporal lobar degeneration, Huntington's disease,HIV-associated dementia, Kennedy's disease, Krabbe's disease, Lewy bodydementia, neuroborreliosis, Machado-Joseph disease (Spinocerebellarataxia type 3), Multiple System Atrophy, multiple sclerosis, narcolepsy,Niemann Pick disease, Parkinson's disease, Pelizaeus-Merzbacher Disease,Pick's disease, primary lateral sclerosis, prion diseases, progressivesupranuclear palsy, Refsum's disease, Sandhoff disease, Schilder'sdisease, subacute combined degeneration of spinal cord secondary toPernicious Anaemia, Spielmeyer-Vogt-Sjogren-Batten disease (also knownas Batten disease), spinocerebellar ataxia, spinal muscular atrophy,Steele-Richardson-Olszewski disease, Tabes dorsalis, toxicencephalopathy.

Accordingly, a temperature-sensitive agent (e.g., atemperature-sensitive therapeutic agent) is administered to a subject soas to reduce or ameliorate symptoms associated with a particulardisorder. Therapeutic endpoints for the treatment of cancer can includea reduction in the size or volume of a tumor, reduction in angiogenesisto the tumor, or reduction in metastasis of the tumor. If the tumor hasbeen removed, another therapeutic endpoint can be regeneration of thetissue or organ removed. Effectiveness of cancer treatment can bemeasured using methods in the art, for example imaging of the tumor ordetecting tumor markers or other indicators of the presence of thecancer. Therapeutic endpoints for the treatment of autoimmune diseasescan include a reduction in the autoimmune response. Effectiveness ofautoimmune disease treatment can be measured using methods in the art,for example measuring of autoimmune antibodies, wherein a reduction insuch antibodies in the treated subject indicates that the therapy issuccessful. Therapeutic endpoints for the treatment of neurodegenerativedisorders can include a reduction in neurodegenerative-related deficits,e.g., an increase in motor, memory or behavioral deficits. Effectivenessof treating neurodegenerative disorders can be measured using methods inthe art, for example by measuring cognitive impairment, wherein areduction in such impairment in the treated subject indicates that thetherapy is successful. Therapeutic endpoints for the treatment ofneurologic injuries can include a reduction in injury-related deficits,e.g., an increase in motor, memory or behavioral deficits. Effectivenessof treating neurologic injuries can be measured using methods in theart, for example by measuring mobility and flexibility, wherein anincrease in such in the treated subject indicates that the therapy issuccessful. Treatment does not require 100% effectiveness. A reductionin the disease (or symptoms thereof) of at least about 10%, about 15%,about 25%, about 40%, about 50%, or greater, for example relative to theabsence of treatment with the agent in human cells, is consideredeffective.

Temperature-sensitive agents (e.g., a temperature-sensitive therapeuticagents) of the present disclosure may also be used to treatatherosclerosis and/or a coronary heart disease in a subject in needthereof, by, for example, administering a temperature-sensitive agent(e.g., a temperature-sensitive therapeutic agent) of the presentdisclosure to the bloodstream of the subject such that the agentintroduces/contacts and increases quality of vascular endothelial cells,thereby treating atherosclerosis and/or a coronary heart disease in thesubject.

Temperature-sensitive agents (e.g., a temperature-sensitive therapeuticagents) of the present disclosure may also be used to provide resistanceto one or more genotoxic agents in one or more human cells and/or asubject in need thereof.

Examples of disorders or diseases that can benefit from administrationof a temperature-sensitive agent (e.g., a temperature-sensitivetherapeutic agent) include those disorders or diseases that areassociated with gene mutation(s), abnormal telomere length, or abnormalepigenetic modification(s). Further examples of disorders or diseasesthat can benefit from administration of a temperature-sensitive agent(e.g., a temperature-sensitive therapeutic agent) include cancer,autoimmune diseases, and diseases in which cell regeneration isbeneficial, such as neurologic injuries or a neurodegenerativedisorders, as well as blindness and deafness.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means including A, or B, or A and B. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control.

Exemplary Embodiments

1. A method for transiently inducing a temperature-sensitive activity ofa temperature-sensitive agent, comprising:

i) incubating one or more cells comprising the temperature-sensitiveagent at a permissive temperature to induce the temperature-sensitiveactivity for a period of time sufficient for the temperature-sensitiveactivity to produce an effect in the one or more cells; and

ii) incubating the one or more cells at a non-permissive temperature,wherein the non-permissive temperature reduces the temperature-sensitiveactivity of the temperature-sensitive agent,

wherein the temperature-sensitive agent comprises a therapeutic agentand the effect comprises a therapeutic effect.

2. The method of embodiment 1, further comprising before step i):contacting the one or more cells with the temperature-sensitive agent.

3. The method of embodiment 2, wherein the one or more cells are at thepermissive temperature when contacted with the temperature-sensitiveagent.

4. The method of any one of embodiments 1-3, further comprisingadministering the one or more cells to a subject in need of thetherapeutic effect.

5. The method of any one of embodiments 1-3, wherein incubating the oneor more cells at a non-permissive temperature comprises administeringthe one or more cells to a subject in need of the therapeutic effect,wherein the subject's body temperature is the non-permissivetemperature.6. The method of embodiment 4 or 5, wherein the one or more cells arefurther incubated at the non-permissive temperature prior toadministering the one or more cells to the subject7. The method of any one of embodiments 1-6, wherein the one or morecells are embryonic stem cells or induced pluripotent stem cells.8. The method of embodiment 7, wherein the therapeutic effect comprisesthe differentiation of said cells into a desired cell type.9. The method of embodiment 8, wherein the desired cell type is selectedfrom the group consisting of a neuron, a glia cell, and an endothelialcell.10. The method of any one of embodiments 2-9, wherein the one or morecells were isolated from the subject before contacting the one or morecells with the temperature-sensitive agent.11. A method for transiently inducing a temperature-sensitive activityof a temperature-sensitive agent in a subject, wherein one or more cellsof the subject comprise the temperature-sensitive agent, wherein thetemperature-sensitive activity of the temperature-sensitive agent isinduced at a permissive temperature, and wherein the permissivetemperature is lower than the body temperature of the subject,comprising:

i) lowering the body temperature of the subject to the permissivetemperature;

ii) maintaining said lowered body temperature for a period of timesufficient for the temperature-sensitive activity to induce an effect inthe subject; and

iii) increasing the body temperature of the subject back to normal bodytemperature.

12. A method for transiently inducing a temperature-sensitive activityof a temperature-sensitive agent in a subject, wherein thetemperature-sensitive activity of the temperature-sensitive agent isinduced at a permissive temperature, and wherein the permissivetemperature is lower than the body temperature of the subject,comprising:

i) lowering the body temperature of the subject to the permissivetemperature;

ii) administering the temperature-sensitive agent to one or more cellsof the subject;

iii) maintaining said lowered body temperature for a period of timesufficient for the temperature-sensitive activity to induce an effect inthe subject; and

iv) increasing the body temperature of the subject back to normal bodytemperature, wherein step (i) is performed before, after, orsimultaneously with step (ii).

13. The method of embodiment 12, wherein the temperature-sensitive agentis administered systemically.

14. The method of embodiment 13, wherein the temperature-sensitive agentis administered intravenously.

15. The method of embodiment 12, wherein the temperature-sensitive agentis administered to a specific tissue or organ of the subject.

16. The method of embodiment 15, wherein the temperature-sensitive agentis administered to the brain and spinal cord by epidural injection.

17. The method of embodiment 15, wherein the temperature-sensitive agentis administered by percutaneous injection into a target organ.

18. The method of embodiment 15, wherein the temperature-sensitive agentis administered by endoscopy with an injection needle catheter into atarget organ.

19. The method of embodiment 15, wherein the temperature-sensitive agentis administered by angiocatheter into a target organ.

20. The method of any one of embodiments 17-19, wherein the target organis selected from the group consisting of the liver, kidneys, skeletalmuscles, cardiac muscles, pancreas, spleen, heart, brain, spinal cord,skin, eye, lung, intestine, thymus, bone marrow, bone, and cartilage.21. The method of embodiment 12, wherein the temperature-sensitive agentis administered by inhalation.22. The method of any one of embodiments 11-21, wherein altering thebody temperature of a subject comprises using a targeted temperaturemanagement (TTM) procedure, wherein the TTM procedure comprisesapplication to the subject of one of the group consisting of a coolingcatheter, a cooling blanket, and ice.23. The method of any one of embodiments 11-22, wherein the subject is amammalian subject, optionally wherein the subject is a human.24. The method of any one of embodiments 11-23, wherein thetemperature-sensitive agent comprises a therapeutic agent, and theeffect comprises a therapeutic effect.25. The method embodiment 24, or any one of embodiments 1-10, whereinthe therapeutic agent is encoded by a coding region of a heterologousnucleic acid of a temperature-sensitive viral vector.26. The method of embodiment 25, wherein said temperature-sensitiveviral vector is selected from the group consisting of a Sendai virus, anAdeno virus, an Adeno-associated virus, a Retrovirus, and an Alphavirus.27. The method of embodiment 26, wherein said temperature-sensitiveviral vector is an Alphavirus.28. The method of embodiment 27, wherein said Alphavirus is selectedfrom the group consisting of a Venezuelan equine encephalitis virus, aSindbis virus, and a Semliki Forrest virus.29. The method of embodiment 26, wherein the temperature-sensitive viralvector is a Sendai virus.30. The method of any one of embodiments 25-29, wherein the therapeuticagent is selected from the group consisting of a non-coding RNA, asiRNA, a shRNA, and an endonuclease editing system.31. The method of any one of embodiments 25-29, wherein the therapeuticagent is a protein.32. The method of embodiment 31, wherein the protein is a transcriptionfactor, optionally wherein the transcription factor is selected from thegroup consisting of human neurogenin-3 (NGN3), and human ETStranslocation variant 2 (ETV2).33. The method of embodiment 31, wherein the protein is a growth factor,optionally wherein the growth factor is selected from the groupconsisting of brain-derived neurotrophic factor (BDNF) and nerve growthfactor (NGF).34. The method of any one of embodiments 25-33, wherein the temperaturesensitive activity comprises replication and transcription of thetemperature-sensitive viral vector.35. The method of embodiment 24, or any one of embodiments 1-10, whereinthe therapeutic agent is encoded by a coding region of atemperature-sensitive self-replicating RNA.36. The method of embodiment 35, wherein said self-replicating RNAcomprises an Alphavirus replicon lacking a viral structural proteincoding region.37. The method of embodiment 36, wherein said Alphavirus is selectedfrom the group consisting of a Venezuelan equine encephalitis virus, aSindbis virus, and a Semliki Forrest virus.38. The method of any one of embodiments 35-37, wherein the therapeuticagent is selected from the group consisting of a non-coding RNA, asiRNA, a shRNA, and an endonuclease editing system.39. The method of any one of embodiments 35-37, wherein the therapeuticagent is a protein.40. The method of embodiment 39, wherein the protein is a transcriptionfactor, optionally wherein the transcription factor is selected from thegroup consisting of human neurogenin-3 (NGN3), and human ETStranslocation variant 2 (ETV2).41. The method of embodiment 39, wherein the protein is a growth factor,optionally wherein the growth factor is selected from the groupconsisting of brain-derived neurotrophic factor (BDNF) and nerve growthfactor (NGF).42. The method of any one of embodiments 35-41, wherein thetemperature-sensitive activity comprises one or both of replication andtranscription of the temperature-sensitive self-replicating RNA.43. The method of any one of embodiments 25-42, wherein the codingregion is operably linked to a promoter.44. The method of any one of embodiments 1-10, wherein the period oftime sufficient for the temperature-sensitive activity to produce atherapeutic effect ranges from about 12 hours to about 12 weeks,optionally wherein the period of time is from 1 to 7 days.45. The method of any one of embodiments 11-43, wherein the period oftime sufficient to induce an effect in the subject is from about 12hours to about 7 days, optionally wherein the period of time is fromabout 12 hours to about 72 hours.46. The method of any one of embodiments 1-45, wherein the permissivetemperature ranges from 30° C. to 36° C. or from 38° C. to 50° C.47. The method of embodiment 46, wherein the permissive temperature is33° C.±0.5° C.48. The method of embodiment 46 or embodiment 47, wherein thenon-permissive temperature is 37° C.±0.5° C.49. The method of any one of embodiments 1-48, wherein the one or morecells are human cells.50. The method of embodiment 49, wherein the one or more human cells areadult stem cells, tissue stem cells, progenitor cells, embryonic stemcells, or induced pluripotent stem cells.51. The method of embodiment 50, wherein the one or more human cells areselected from the group consisting of hematopoietic stem cells,mesenchymal stem cells, adipose stem cells, neuronal stem cells, andgerm stem cells.52. The method of embodiment 49, wherein the one or more human cells aresomatic cells, mature cells, or differentiated cells.53. The method of embodiment 52, wherein the one or more human cells areselected from the group consisting of epidermal cells, fibroblasts,lymphocytes, hepatocytes, epithelial cells, myocytes, chondrocytes,osteocytes, adipocytes, cardiomyocytes, pancreatic cells, pancreatic pcells, keratinocytes, erythrocytes, peripheral blood mononuclear cells(PBMC), neurons, glia cells, neurocytes, astrocytes, germ cells, spermcells, and oocytes.54. The method of embodiment 49, wherein the one or more human cells arehuman bone marrow cells.55. The method of embodiment 54, wherein the human bone marrow cells areselected from the group consisting of hematopoietic stem cells,mesenchymal stem cells, and endothelial stem cells.56. The method of embodiment 55, wherein the hematopoietic stem cellsare CD34+.57. The method of any one of embodiments 25-56, wherein thetemperature-sensitive viral vector or the temperature-sensitiveself-replicating RNA comprises a nonstructural protein coding regionwith an insertion of 12-18 nucleotides, wherein the insertion results inexpression of a nonstructural Protein 2 (nsP2=helicase proteinase)comprising from 4 to 6 additional amino acids between beta sheet 5 andbeta sheet 6 of the nsP2, optionally wherein the additional amino acidsresult in temperature-sensitivity of the viral vector or theself-replicating RNA.58. The method of embodiment 57, wherein the additional amino acidscomprise one sequence selected from the group consisting of SEQ ID NO:38(GCGRT), SEQ ID NO:39 (TGAAA), and SEQ ID NO:40 (LRPHP).59. The method of embodiment 57, wherein the additional amino acidscomprise the sequence of SEQ ID NO:39 (TGAAA).60. The method of embodiment 59, wherein the amino acid sequence of theNsP2 comprises one sequence selected from the group consisting of SEQ IDNOs:29-36.61. A temperature-sensitive agent, wherein the agent is atemperature-sensitive viral vector or a temperature-sensitiveself-replicating RNA comprising a nonstructural protein coding regionwith an insertion of 12-18 nucleotides, wherein the insertion results inexpression of a nonstructural Protein 2 (nsP2=helicase proteinase)comprising from 4 to 6 additional amino acids between beta sheet 5 andbeta sheet 6 of the nsP2, optionally wherein the additional amino acidsresult in temperature-sensitivity of the viral vector or theself-replicating RNA.62. The temperature-sensitive agent of embodiment 61, wherein theadditional amino acids comprise one sequence selected from the groupconsisting of SEQ ID NO:38 (GCGRT), SEQ ID NO:39 (TGAAA), and SEQ IDNO:40 (LRPHP).63. The temperature-sensitive agent of embodiment 61, wherein theadditional amino acids comprise the sequence of SEQ ID NO:39 (TGAAA).64. The temperature-sensitive agent of embodiment 63, wherein the aminoacid sequence of the NsP2 comprises one sequence selected from the groupconsisting of SEQ ID NOs:29-36.65. The temperature-sensitive agent of any one of embodiments 61-64,wherein the agent is a temperature-sensitive Alphavirus vector.66. The temperature-sensitive agent of any one of embodiments 61-64,wherein the agent is a temperature-sensitive self-replicating RNAcomprising an Alphavirus replicon lacking a viral structural proteincoding region.67. The temperature-sensitive agent of embodiment 65 or embodiment 66,wherein the Alphavirus is selected from the group consisting of aVenezuelan equine encephalitis virus, a Sindbis virus, and a SemlikiForrest virus.68. The temperature-sensitive agent of embodiment 65 or embodiment 66,wherein the Alphavirus is a Venezuelan equine encephalitis virus.69. A method for transiently inducing a temperature-sensitive activityof a temperature-sensitive agent (ts-agent) in a subject, wherein one ormore cells at or near the surface of the subject's body comprise thets-agent, wherein the temperature-sensitive activity of the ts-agent isinduced at a permissive temperature, and wherein the permissivetemperature is the surface body temperature of the subject, comprising:

i) maintaining the surface body temperature of the subject at thepermissive temperature for a period of time sufficient for thetemperature-sensitive activity to induce an effect in the subject; and

ii) increasing the surface body temperature of the subject to anon-permissive temperature for a period of time sufficient for thetemperature-sensitive activity to cease in the subject.

79. A method for transiently inducing a temperature-sensitive activityof a temperature-sensitive agent (ts-agent) in a subject, wherein thetemperature-sensitive activity of the ts-agent is induced at apermissive temperature, and wherein the permissive temperature is thesurface body temperature of the subject, comprising:

i) administering the ts-agent to one or more cells at or near thesurface of the subject's body; and

ii) maintaining the surface body temperature of the subject at thepermissive temperature for a period of time sufficient for thetemperature-sensitive activity to induce an effect in the subject.

71. The method of embodiment 70, further comprising iii) increasing thesurface body temperature of the subject to a non-permissible temperaturefor a period of time sufficient for the temperature-sensitive activityto cease in the subject.

72. The method of embodiment 70 or embodiment 71, wherein thetemperature-sensitive agent is administered intradermally orsubcutaneously.

73. The method of embodiment 70 or embodiment 71, wherein thetemperature-sensitive agent is administered intramuscularly.

74. The method of any one of embodiments 69-73, wherein thenon-permissive temperature is above 36° C., and the permissivetemperature is below 36° C., optionally wherein the permissivetemperature is from about 31° C. to about 34° C., or about 33° C.±0.5°C., and the non-permissive temperature is 37° C.±0.5° C.75. The method of any one of embodiments 69-74, wherein a pharmaceuticalagent is encoded by a coding region of the ts-agent or the ts-agentcomprises a pharmaceutical agent, and the effect comprises apharmaceutical effect, optionally wherein the pharmaceutical agent is atherapeutic agent and the pharmaceutical effect is a therapeutic effect,or wherein the pharmaceutical agent is a prophylactic agent and thepharmaceutical effect is a prophylactic effect.76. The method of any one of embodiments 69-75, wherein the ts-agent isa temperature-sensitive viral vector and the temperature-sensitiveactivity comprises replication and transcription of thetemperature-sensitive viral vector.77. The method of embodiment 76, wherein the temperature-sensitive viralvector is selected from the group consisting of a Sendai virus, an Adenovirus, an Adeno-associated virus, a Retrovirus, and an Alphavirus.78. The method of embodiment 76, wherein the temperature-sensitive viralvector is an Alphavirus, optionally wherein the Alphavirus is selectedfrom the group consisting of a Venezuelan equine encephalitis virus, aSindbis virus, and a Semliki Forrest virus.79. The method of embodiment 76, wherein the temperature-sensitive viralvector is a Sendai virus.80. The method of any one of embodiments 69-75, wherein the ts-agent isa temperature-sensitive self-replicating RNA and thetemperature-sensitive activity comprises one or both of replication andtranscription of the temperature-sensitive self-replicating RNA.81. The method of embodiment 80, wherein the self-replicating RNAcomprises an Alphavirus replicon lacking an Alphavirus viral structuralprotein coding region.82. The method of embodiment 81, wherein the Alphavirus is selected fromthe group consisting of a Venezuelan equine encephalitis virus, aSindbis virus, and a Semliki Forrest virus.83. The method of embodiment 81, wherein the Alphavirus is a Venezuelanequine encephalitis virus.84. The method of any one of embodiments 69-83, wherein thepharmaceutical agent is selected from the group consisting of anon-coding RNA, a siRNA, a shRNA, and an endonuclease editing system.85. The method of any one of embodiments 69-83, wherein thepharmaceutical agent comprises a protein.86. The method of embodiment 85, wherein the protein comprises anantigen of a pathogen.87. The method of embodiment 86, wherein the pathogen comprises one ormore of a virus, a bacterium, a protozoan, and a fungus.88. The method of embodiment 86 or embodiment 87, wherein the antigencomprises a surface protein or fragment thereof of the pathogen.89. The method of any one of embodiments 69-88, wherein the period oftime sufficient for the temperature-sensitive activity to produce aneffect ranges from about 12 hours to about 12 weeks, optionally whereinthe period of time is from 1 to 7 days.90. The method of any one of embodiments 69-88, wherein the period oftime sufficient to induce an effect in the subject is from about 12hours to about 7 days, optionally wherein the period of time is fromabout 12 hours to about 72 hours.91. An immunogenic composition for stimulating an immune responseagainst a pathogen in a subject, comprising an excipient and atemperature-sensitive agent (ts-agent), wherein the ts-agent is atemperature-sensitive viral vector or a temperature-sensitiveself-replicating RNA encoding an antigen of the pathogen, and whereinthe ts-agent is capable of expressing the antigen at a permissivetemperature but not at a non-permissive temperature.92. The composition of embodiment 91, wherein the pathogen comprises oneor more of a virus, a bacterium, a protozoan, and a fungus.93. The composition of embodiment 91 or embodiment 92, wherein theantigen comprises a surface protein or fragment thereof of the pathogen.94. The composition of embodiment 93, wherein the pathogen is a virus,and the virus is different than the viral vector.95. The composition of embodiment 94, wherein the virus is a coronavirusand the antigen comprises a spike protein or fragment thereof of thecoronavirus.96. The composition of embodiment 95, wherein the coronavirus is2019-nCoV and the antigen comprises a receptor-binding domain (RBD) ofthe 2019-nCoV.97. The composition of embodiment 96, wherein the amino acid sequence ofthe RBD comprises SEQ ID NO:44, or the amino acid sequence at least 75%,85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:44.98. The composition of embodiment 95, wherein the coronavirus is2019-nCoV and the antigen comprises an extracellular region of the spikeprotein comprising the amino acid sequence of residues 16-1213 of SEQ IDNO:41, or the amino acid sequence at least 75%, 85%, 90%, 95%, 96%, 97%,98% or 99% identical to SEQ ID NO:41.99. The composition of any one of embodiments 91-98, wherein thenon-permissive temperature is above 36° C., and the permissivetemperature is below 36° C., optionally wherein the permissivetemperature is from about 31° C. to about 34° C., or about 33° C.±0.5°C., and the non-permissive temperature is 37° C.±0.5° C.100. The composition of any one of embodiments 91-99, wherein thets-agent is a temperature-sensitive self-replicating RNA.101. The composition of embodiment 100, wherein the self-replicating RNAcomprises an Alphavirus replicon lacking a viral structural proteincoding region.102. The composition of embodiment 101, wherein the Alphavirus isselected from the group consisting of a Venezuelan equine encephalitisvirus, a Sindbis virus, and a Semliki Forrest virus.103. The composition of embodiment 101, wherein the Alphavirus is aVenezuelan equine encephalitis virus.104. The composition of any one of embodiments 91-99, wherein thets-agent is a temperature-sensitive viral vector.105. The composition of embodiment 104, wherein the viral vector isselected from the group consisting of a Sendai virus, an Adeno virus, anAdeno-associated virus, a Retrovirus, and an Alphavirus.106. The composition of embodiment 105, wherein the Alphavirus isselected from the group consisting of a Venezuelan equine encephalitisvirus, a Sindbis virus, and a Semliki Forrest virus.107. The composition of embodiment 105, wherein the viral vector is aSendai virus.108. The method of any one of embodiments 69-90 or the composition ofany one of embodiments 91-107, wherein the subject is a mammaliansubject, optionally wherein the subject is a human.109. The composition of any one of embodiments 91-108, wherein thepathogen is a mammalian pathogen, optionally wherein the pathogen is ahuman pathogen.110. A method for stimulating an immune response against a pathogen in amammalian subject, comprising administering the immunogenic compositionof embodiment 109 to a mammalian subject so as to stimulate an immuneresponse against the antigen in the mammalian subject, optionallywherein the mammalian subject is a human subject.111. The method of embodiment 110, wherein the immunogenic compositionis administered:

i) intradermally or subcutaneously; or

ii) intramuscularly.

112. The method of embodiment 110, wherein the immunogenic compositionis administered intranasally.

EXAMPLES

Abbreviations: Aura (Aura virus); BFV (Barmah Forest virus); GFP (greenfluorescent protein); GOI (gene of interest); IRES (internal ribosomeentry site); LUC (luciferase); OD (optical density); ONNV (O'nyong-nyongvirus); RBD (receptor binding domain); RRV (Ross River virus); SeV(Sendai virus); SeVts (temperature-sensitive Sendai virus); SFV (SemlikiForest virus); shRNA (short hairpin RNA); SINV (Sindbis virus); srRNA(self-replicating RNA); ts (temperature-sensitive); ts-agent(temperature-sensitive-agent); VEEV (Venezuelan equine encephalitisvirus); and WEEV (Western equine encephalitis virus).

The following examples are provided to illustrate certain particularfeatures and/or embodiments. The examples are not intended to limit thedisclosure as claimed.

Example 1: Temperature-Sensitive Agents

This example describes a temperature-sensitive agent (ts-agent) thatfunctions at a lower or higher temperature than normal body temperature,but does not function, or shows reduced functionality, at normal bodytemperature. The ts-agents are suitable for use in ex vivo, semi invivo, and in vivo therapies. Temperature-sensitive viral vectors andself-replicating RNAs are engineered to express a gene of interest(GOI), a short hairpin RNA (shRNA), a long non-coding RNA, and/or othergenetic element(s). For instance, proteins with temperature-sensitivemutations, are functional at a lower temperature (e.g., at 30° C.), butare not functional at a normal body temperature (e.g., at 37° C.).Unless otherwise specified, normal body temperature is normal human bodytemperature of 37° C.±0.5° C.

Example 2: Temperature-Sensitive Sendai Virus Vectors (SeVts)

This example describes temperature-sensitive Sendai virus vectors(SeVts), which can be used for temperature-specific gene expression.Sendai virus vectors are based on the Sendai virus, a single-strandedRNA virus of the Paramyxovirus subfamily. SeV18/TS15ΔF is atemperature-sensitive Sendai virus vector, which allows for viralreplication and gene expression when held at 32-35° C. However, viralreplication ceases at non-permissive temperatures of 37° C. and above(Ban et al., PNAS 2011).

Example 3: Temperature-Sensitive Self-Replicating RNAs (srRNAs)

This example describes the finding that a mutation in nsP2 proteinencoded in a Venezuelan Equine Encephalitis Virus (VEEV) Vector exhibitstemperature sensitivity. The temperature-sensitive system permitsexpression of a gene of interest (GOI) at 30° C.-33° C., but not at 37°C. and above. The srRNA vector permits higher expression of the GOI thana synthetic RNA encoding the GOI. The expression of the GOI is turnedoff, when the temperature is shifted to 37° C. (e.g., a non-permissivetemperature). A specific temperature-sensitive mutation (mutation 2)described below is in a well-conserved region of among Alphaviruses.Compared to Sendai Virus Vectors (SeVts), srRNAts may be more attractivefor some applications, as srRNAts can be utilized in non-viral RNAexpression systems.

Materials and Methods

Cell Culture

A human Adipose Stem Cell-derived iPS cell line (ADSC-iPS cells) waspurchased from System Biosciences (Palo Alto, Calif.). Cells wereroutinely maintained as undifferentiated human pluripotent stem cells(hPSCs) according to the standard hPSC culture method. Briefly, cellswere cultured in StemFit basic02 (Ajinomoto, Japan) supplemented with100 ng/ml FGF2. Further, cells were cultured on cell culture dishescoated with a laminin-511 substrate (iMatrix-511, Nippi, Japan).

VEEV Vector

A VEEV vector plasmid was assembled using synthesized DNA fragmentsbased on the publicly available sequence information (T7-VEE-IRES-Puro,herein after “srRNA1 wt”). Per Yoshioka et al., 2013, the VEEV vectorbackbone was originally derived as in Petrakova et al., 2005. 7480candidate sequences identified by insertional mutagenesis and massivelyparallel sequencing (Beitzel et al., 2010) were used to derive potentialtemperature-sensitive mutants. The original large-scale screen wasperformed by transposon-mediated insertion of 15 bp into the VEEV genome(FIG. 1A). Subsequently, a large number of 15-bp-insertion VEEV mutantsthat were able to proliferate at 30° C. or 40° C. were isolated.Although these data provided initial mutants for further research, itwas not known whether these sequences exhibit temperature sensitivity,such as permissiveness at 32° C. or 33° C. and non-permissiveness at 37°C. Three mutant sequences—Mutant 1 (ts1, FIG. 1B), Mutant 2 (ts2, FIG.1C), and Mutant 3 (ts3, FIG. 1D) were selected from a total of 7480candidate mutant sequences (Data Set Si from Beitzel et al., 2010).These mutant DNA fragments (FIG. 2) were synthesized and cloned into theVEEV vector and named srRNA1ts1 (mutant1), srRNA1ts2 (mutant 2),srRNA1ts3 (mutant 3). A mutant 4 was designed, which includes the5′-region of virus sequence (5′-UTR and a part of N-terminal proteinsequence of RNA-dependent RNA polymerase known to include a 51-ntconserved sequence element (CSE)). In this case, nucleotides weresystematically changed to less thermo-stable variants (e.g., G→A), whilemaintaining the amino acid sequences (FIG. 3). The sequence of thisregion in srRNA1ts2 was replaced to generate srRNA1ts4 (i.e., containingboth mutant 4 and mutant 2). Synthetic RNAs were produced from thesevectors according to Yoshioka et al., 2013.

Results

Assessing Temperature-Sensitivity of srRNA1ts2-GFP and srRNA1ts3-GFP at30° C., 32° C., and 37° C.

ADSC-iPSC cells were plated on a 24-well plate at a density of 80,000cells/well. After 24 hours, cells were transfected with srRNA 1 wt-GFP,srRNA1ts2-GFP, or srRNA1ts3-GFP. For transfection, each well of a24-well plate was treated with, 0.5 μg synthetic RNA (srRNA) mixed with1 μl of JetMessenger (Polyplus) transfection reagent at a final volumeof 50 μl. After adding the transfection complex to the cells, 450 μl ofculture media was added. The cells were incubated at either 30° C., 32°C., or 37° C. At 6 hours after transfection, the medium was changed toremove the transfection complex. The phase-contrast and fluorescentimages were taken at 20 hours and 48 hours. FIG. 4A shows that wild type(srRNA1wt-GFP) strongly expressed GFP at 37° C., but only weaklyexpressed GFP at both 30° C. and 32° C. By contrast, mutant 2(srRNA1ts2-GFP) expressed GFP at 30° C. and 32° C., but not at 37° C.Mutant 3 (srRNA1ts3-GFP) expressed GFP at 30° C. and 32° C., but alsoexpressed GFP at 37° C. Based on these results, mutant 2 was selectedfor further development. As expected, srRNA showed much higherexpression of GFP, compared to the GFP expression levels that wereachieved by a single transfection of synthetic mRNA encoding the GFP(FIG. 4B).

Assessing Temperature-Sensitivity of srRNA1ts1-GFP and srRNA1Ts2-GFP at32° C.

ADSC-iPSC cells were plated on a 24-well plate at the density of 50,000cells/well. After 24 hours, cells were transfected with srRNA 1 wt-GFP,srRNA1ts2-GFP, or srRNA1ts1-GFP. For transfection, each well of a24-well plate was treated with, 0.5 μg synthetic RNA (srRNA) mixed with1 μl of JetMessenger (Polyplus) transfection reagent at a final volumeof 50 μl. After adding the transfection complex to the cells, 450 μl ofculture media was added. The cells were incubated at 32° C. At 6 hoursafter transfection, the medium was changed to remove the transfectioncomplex. The phase-contrast and fluorescent images were taken at 24, 48,72, 96, 120, 144, 168, 192, 240, and 288 hours.

FIG. 5 shows the results. The GFP expression from wild type (srRNA1wt-GFP) started at 24 hours and continued until the end of theobservation period (at 288 hours), but was very weak throughout the timecourse. By contrast, the GFP expression from a mutant 2 (srRNA1ts2-GFP)was very strong throughout the time course. Mutant 1 (srRNA1ts1-GFP) didnot express GFP at all (based on observation at 24 hours and 168 hours).Based on these results, mutant 2 was selected for further development.

Assessing Temperature-Sensitivity of srRNA1ts2-GFP and srRNA1ts4-GFP at32° C., 33° C., and 37° C.

ADSC-iPSC cells were plated on a 24-well plate at the density of 50,000cells/well. After 24 hours, cells were transfected with srRNA 1ts2-GFP,or srRNA1ts4-GFP. For transfection, each well of a 24-well plate wastreated with, 0.5 μg synthetic RNA (srRNA) mixed with 1 μl ofJetMessenger (Polyplus) transfection reagent at a final volume of 50 μl.After adding the transfection complex to the cells, 450 μl of culturemedia was added. The cells were incubated at either 32° C., 33° C., or37° C. At 6 hours after transfection, the medium was changed to removethe transfection complex. The phase-contrast and fluorescent images weretaken at 20, 48, and 96 hours.

FIG. 6 shows the results. At 32° C. and 33° C., the GFP expression frommutant 2 (srRNA1ts2-GFP) started as early as 20 hours, but significantlyincreased at 48 hours, and further increased at 96 hours. The expressionof GFP was stronger at 33° C. than at 32° C. Consistent with theexperiments above, GFP was not expressed at all at 37° C. ThesrRNA1ts4-GFP (containing both a mutant 2 and mutant 4) showed a similartemperature profile to srRNA1ts2-GFP, but the GFP expression was muchweaker overall. Based on these results, mutant 2 was selected forfurther development.

Assessing Temperature-Sensitivity of srRNA1ts2-GFP at 32° C.

ADSC-iPSC cells were plated on a 24-well plate at the density of 80,000cells/well. After 24 hours, cells were transfected with srRNA 1ts2-GFP.For transfection, each well of a 24-well plate was treated with, 0.5 μgsynthetic RNA (srRNA) mixed with 1 μl of JetMessenger (Polyplus)transfection reagent at a final volume of 50 μl. After adding thetransfection complex to the cells, 450 μl of culture media was added.The cells were incubated at 32° C. At 6 hours after transfection, themedium was changed to remove the transfection complex. The medium waschanged every day. The srRNA1ts2-GFP vector contains a puromycinN-acetyltransferase (pac) selection gene inserted after the “IRES”sequence, and thus, can be selected using puromycin. The experimentswere done in the absence (upper panel) or presence (lower panel) of 1μg/ml of puromycin. For the cells with puromycin selection, puromycinwas added at 48 hours and 72 hours. The phase-contrast and fluorescentimages were taken at 24, 48, 72, 96, 144, 168, 192 hours.

FIG. 7 shows the results. At 32° C., the GFP expression fromsrRNA1ts2-GFP started as early as 24 hours, but significantly increasedat 48 hours, and peaked at 72 hours and 96 hours. The expression of GFPcontinued until the end of observation period (at 192 hours). Theexpression pattern of GFP did not seem to be altered by the addition ofpuromycin.

Assessing Temperature-Sensitivity of srRNA1ts2-GFP

Switched from 32° C. to 37° C. after 24 Hours.

ADSC-iPSC cells were plated on a 24-well plate at the density of 80,000cells/well. After 24 hours, cells were transfected with srRNA 1ts2-GFP.For transfection, each well of a 24-well plate was treated with, 0.5 μgsynthetic RNA (srRNA) mixed with 1 μl of JetMessenger (Polyplus)transfection reagent at a final volume of 50 μl. After adding thetransfection complex to the cells, 450 μl of culture media was added.The cells were incubated at 32° C. At 6 hours after transfection, themedium was changed to remove the transfection complex. The medium waschanged every day. The srRNA1ts2-GFP vector contains a puromycinN-acetyltransferase (pac) selection gene inserted after the “IRES”sequence, and thus, can be selected using puromycin. The experimentswere done in the absence (upper panel) or presence (lower panel) of 1μg/ml of puromycin. For the cells with puromycin selection, puromycinwas added at 48 hours and 72 hours. To test the effects of temperatureshift, the cell cultures were transferred to a CO₂ incubator maintainedat 37° C. at 24 hours (24 hours after the transfection). Thephase-contrast and fluorescent images were taken at 24, 48, 72, 96, 144,168, 192 hours.

FIG. 8 shows the results. At 32° C., the GFP expression fromsrRNA1ts2-GFP started as early as 24 hours, but continued to increaseeven after the switching of temperature to 37° C. at 24 hours. Theexpression of GFP peaked at 48, and then, started to decrease. By 96hours, the GFP expression became very weak and by 144 hours the GFPexpression could not be detected any more. Subsequently, there was noGFP expression until the end of observation period at 192 hours. Thus,the expression of the GOI (represented here by GFP) was rapidly turnedoff, when the temperature shifted from 33° C. (a permissive temperature)to 37° C. (a non-permissive temperature). The expression pattern of GFPdid not seem to be altered by the addition of puromycin.

Assessing Temperature-Sensitivity of srRNA1ts2-GFP

Switched from 32° C. to 37° C. after 48 Hours.

ADSC-iPSC cells were plated on a 24-well plate at the density of 80,000cells/well. After 24 hours, cells were transfected with srRNA 1ts2-GFP.For transfection, each well of a 24-well plate was treated with, 0.5 μgsynthetic RNA (srRNA) mixed with 1 μl of JetMessenger (Polyplus)transfection reagent at a final volume of 50 μl. After adding thetransfection complex to the cells, 450 μl of culture media was added.The cells were incubated at 32° C. At 6 hours after transfection, themedium was changed to remove the transfection complex. The medium waschanged every day. The srRNA1ts2-GFP vector contains a puromycinN-acetyltransferase (pac) selection gene inserted after the “IRES”sequence, and thus, can be selected using puromycin. The experimentswere done in the absence (upper panel) or presence (lower panel) of 1μg/ml of puromycin. For the cells with puromycin selection, puromycinwas added at 48 hours and 72 hours. To test the effects of temperatureshift, the cell cultures were transferred to a CO₂ incubator maintainedat 37° C. at 48 hours (48 hours after the transfection). Thephase-contrast and fluorescent images were taken at 24, 48, 72, 96, 144,168, 192 hours.

FIG. 9 shows the results. At 32° C., the GFP expression fromsrRNA1ts2-GFP started as early as 24 hours and further increased at 48hours. The expression of GFP continued until 96 hours even after theswitching of temperature to 37° C. at 48 hours. But the GFP expressionstarted to decrease from 72 hours and by 96 hours the GFP expressionbecame very weak. By 144 hours the GFP expression was barely detectedand completely turned off by 192 hours. Thus, the expression of the GOI(represented here by GFP) was rapidly turned off, when the temperatureshifted from 33° C. (a permissive temperature) to 37° C. (anon-permissive temperature). The expression pattern of GFP did not seemto be altered by the addition of puromycin.

Assessing Temperature-Sensitivity of srRNA1ts2-GFP

Switched from 32° C. to 37° C. after 72 Hours.

ADSC-iPSC cells were plated on a 24-well plate at the density of 80,000cells/well. After 24 hours, cells were transfected with srRNA 1ts2-GFP.For transfection, each well of a 24-well plate was treated with, 0.5 μgsynthetic RNA (srRNA) mixed with 1 μl of JetMessenger (Polyplus)transfection reagent at a final volume of 50 μl. After adding thetransfection complex to the cells, 450 μl of culture media was added.The cells were incubated at 32° C. At 6 hours after transfection, themedium was changed to remove the transfection complex. The medium waschanged every day. The srRNA1ts2-GFP vector contains a puromycinN-acetyltransferase (pac) selection gene inserted after the “IRES”sequence, and thus, can be selected using puromycin. The experimentswere done in the absence (upper panel) or presence (lower panel) of 1μg/ml of puromycin. For the cells with puromycin selection, puromycinwas added at 48 hours and 72 hours. To test the effects of temperatureshift, the cell cultures were transferred to a CO₂ incubator maintainedat 37° C. at 72 hours (72 hours after the transfection). Thephase-contrast and fluorescent images were taken at 24, 48, 72, 96, 144,168, 192 hours.

FIG. 10 shows the results. At 32° C., the GFP expression fromsrRNA1ts2-GFP started as early as 24 hours and further increased at 48hours. The expression of GFP continued until 96 hours even after theswitching of temperature to 37° C. at 48 hours. But the GFP expressionstarted to decrease from 72 hours and by 144 hours the GFP expressionbecame very weak. By 168 hours the GFP expression was barely detectedand completely turned off by 192 hours. Thus, the expression of the GOI(represented here by GFP) was rapidly turned off, when the temperatureshifted from 33° C. (a permissive temperature) to 37° C. (anon-permissive temperature). The expression pattern of GFP did not seemto be altered by the addition of puromycin.

Assessing Temperature-Sensitivity of srRNA1ts2-GFP in Fibroblast Cells

Human newborn dermal fibroblast cells (HDFn at passage 20) were platedon a 24-well plate at the density of 10,000 cells/well. After 24 hours,cells were transfected with srRNA1wt-GFP. Transfection of srRNA1wt-GFP(0.5 μg synthetic RNA) was carried out by using either JetMessenger(Polyplus) transfection reagent or Lipofectamine MessengerMax(Thermo-Fisher). The cells were incubated at 37° C. To see the effect ofB18R, which is known to repress interferon responses, the transfectionand cell culture were carried out in the absence (upper panel) orpresence (lower panel) of 200 ng/ml B18R. The medium was changed everyday. The phase-contrast and fluorescent images were taken at 0, 24, 48,and 96 hours.

FIG. 11 shows the results. In the absence of B18R, almost no expressionof GFP was detected. By contrast, in the presence of B18R, the GFPexpression from srRNA1 wt-GFP started as early as 24 hours and continueduntil 48 hours and 72 hours. The expression of GFP was strong in theGFP+ cells, but the frequency of GFP+ cells was not high. This was mostlikely due to the low transfection efficiency of srRNA1 wt-GFP on humanprimary fibroblast cells.

Aligning Amino Acid Sequences of Alphavirus Family Corresponding toMutant 2 (Ts2)

As shown in FIG. 12, the structure of nsP2 proteins of Alphavirus, evenat the amino acid level, is well conserved among family members. Basedon the 3D structural model (Russo et al., 2006), the protein region,where the 5 amino acids SEQ ID NO:39 (TGAAA) are inserted in the mutant2, is a turning point between two beta-sheet structures, which is alsowell conserved among Alphavirus family members. Therefore, it is highlylikely that the temperature-sensitivity of mutant 2 is transferable toother Alphavirus family members, including Aura (Aura virus), WEEV(Western equine encephalitis virus), BFV (Barmah Forest virus), ONNV(O'nyong-nyong virus), RRV (Ross River virus), SFV (Semliki Forestvirus), and SINV (Sindbis virus). Suitable locations for insertions innsP2 of various Alphaviruses for conferring temperature-sensitivity arelisted in Table 3-1.

TABLE 3-1 Alphavirus nsP2 Sequences and Insertion Sites Insertion Site(:) NCBI amino acids Alphavirus Accession No. positions Venezuelanequine encephalitis virus NP_740697  586:587 Aura virus NP_819011 596:597 Western equine encephalitis virus CAA52868  683:684 BarmahForest virus NP_818996  588:589 Onyong-nyong virus AAC97204 1123:1124Ross River virus NP_740679  587:588 Semliki Forest virus NP_4634571125:1126 Sindbis virus AF492770_1 1137:1138

Example 4: Temperature-Sensitive Antibodies

This example describes temperature-sensitive antibodies. An antibodythat functions at a permissive temperature (e.g., 32° C.) and does notfunction or shows reduced function at a non-permissive temperature(e.g., 37° C.) is engineered by insertion or substitution of amino acidsequences. A temperature-sensitive antibody can be produced by insertinga linker oligonucleotide encoding the temperature-sensitive helix-coiltransition peptide (-Glu-Ala-Ala-Ala-Lys-, set forth as SEQ ID NO:37),as described (Kamihara and Iijima, 2000; Merutka and Stellwagen, 1990).In this way, an engineered antibody can be produced, which functions ata permissive temperature (e.g. 32° C.), but does not function at anon-permissive temperature (e.g., 37° C.). Alternatively, the antibodyDNA sequence of animals naturally living in a low temperatureenvironment (e.g., Atlantic salmon and shrimp) can be used, as theseantibodies are optimally functioning at a permissive temperature (at lowtemperature), but show reduced functionality at a non-permissivetemperature (e.g., 37° C.).

Example 5: Temperature-Sensitive Proteins

This example describes temperature-sensitive proteins. Such proteinsfunction at a permissive temperature (e.g., 32° C.) and do not functionor show low function at a non-permissive temperature (e.g., 37° C.).Temperature-sensitive proteins are engineered by substituting amino acidsequences. Alternatively, temperature-sensitive proteins obtained fromanimals naturally living in a low temperature environment (e.g.,Atlantic salmon and shrimp) can be used, as these proteins are optimallyfunctioning at a permissive temperature (at low temperature), but showreduced functionality at a non-permissive temperature (e.g., 37° C.)(e.g., shrimp alkaline phosphatase).

Example 6: Temperature-Sensitive RNAs

This example describes temperature-sensitive RNA molecules. RNAmolecules include, but are not limited to, mRNA, a precursor of mRNA,non-coding RNA, siRNA, and shRNA. Temperature-sensitive RNAs function ata permissive temperature (e.g., 32° C.) and do not function or show lowfunction at a non-permissive temperature (e.g., 37° C.).Temperature-sensitive RNAs are engineered by systematically changing thenucleotides of RNA molecules to less thermo-stable variants (e.g., G→A),while ensuring that the functional properties of the RNAs aremaintained. Further, the difference in thermostability of the nucleotidepairs, induced by a shift in temperature, changes the secondarystructure of the RNAs.

Example 7: Ex Vivo Treatment of Cells with Temperature-Sensitive Agents

This example demonstrates a method for transiently delivering an RNA orprotein to cells ex vivo (FIG. 13). A temperature-sensitive therapeuticagent can be any of the temperature-sensitive therapeutic agentsdisclosed herein. Ts-agents such as srRNAs or Sendai virus vector arefunctional at a permissive temperature (e.g., 33° C.), butnon-functional at a non-permissive temperature (e.g., 37° C.). Targetcells treated with the ts-agent are cultured ex vivo at a permissivetemperature for a certain duration (e.g., 3 days), and then are culturedat a non-permissive temperature for a certain duration (e.g., 10 days).Levels of RNAs (or proteins translated from the RNAs) of a GOI increaseat a permissive temperature and reach a high level. After switching to anon-permissive temperature, expected levels of RNAs gradually decreaseand subsequently reach to a non-expression level (FIG. 13).

Example 8: Ex Vivo Therapeutic Use of Temperature-Sensitive Agents

This example demonstrates a method for transiently delivering an RNA orprotein to cells ex vivo (FIG. 14 and FIG. 15). Ts-agents such as srRNAsor Sendai virus vector are functional at a permissive temperature (e.g.,33° C.), but non-functional at a non-permissive temperature (e.g., 37°C.: a human body temperature). Typically, target cells are taken from apatient (autologous cell transplants; FIG. 14), but it is also possibleto use target cells isolated from a donor (allogenic cell transplant;FIG. 15). For instance, the target cells may be isolated by usingantibody-conjugated magnetic beads. Target cells are incubated with thets-agent ex vivo at a permissive temperature, e.g., at 33° C. for acertain duration, e.g., 24 hours. Levels of RNAs (or proteins translatedfrom the RNAs) of a GOI increase at a permissive temperature reach ahigh level. After the therapeutic effect is induced, the cells aretransplanted back to the patient in order to treat the patient. Theactivity of the temperature-sensitive therapeutic agent is not inducedat the subject's normal body temperature (i.e. normal body temperatureis a non-permissive temperature). The degradation of thetemperature-sensitive therapeutic agent begins after the therapeuticeffect is induced, and eventually the temperature-sensitive therapeuticagent is completely degraded. The body temperature is maintained at orabove 37° C. for the lifetime of the patient, and thus, the ts-agent isnot reactivated and cells other than the target cells will not betreated with the ts-agent.

Mobilized Human Peripheral Blood Cells

Human blood cells isolated from a patient's, or donor's, bone marrow orperipheral blood are treated with ts-agents ex vivo at a permissivetemperature. After injection of G-CSF or other mobilizing agents, humanwhite blood cells are collected from peripheral blood by an apheresismachine (e.g., COBE Spectra). The white blood cells collected aftermobilization from bone marrow contain granulocytes, monocytes,lymphocytes, dendritic cells, mesenchymal stem cells (MSCs), vascularendothelial cells (VECs), and CD34+ hematopoietic/progenitor cells. Thetreatment of these cells with ts-agents is conducted ex vivo, ideally,using a functionally closed system such as Miltenyi's CliniMacs Prodigy,at a functional temperature (e.g., 33° C.) for a certain duration (a fewhours to a few weeks). Subsequently, the treated cells are infused intopatients at a non-permissive temperature (37° C.). The ts-agents, cellscontaining the ts-agents, or the product of ts-agents do not function inthe patient's body.

Human CD34+ Hematopoietic Stem/Progenitor Cells

Human CD34+ hematopoietic stem/progenitor cells are isolated from themobilized human peripheral blood cells or bone marrow cells by antibody(against CD34)-conjugated magnetic beads and used as target cells aretreated with ts-agents ex vivo at a permissive temperature. Aftertreatment with a ts-agent, human CD34+ cells are infused into apatient's body and engraft in the patient's bone marrow. These cellswill eventually produce all the blood cells in the patient's body, andthus, are a suitable target for a variety of diseases.

Any Human Cells Including Tissue Stem Cells

Any human cells isolated from patient or donor and used as target cellsare treated with ts-agents ex vivo at a permissive temperature. Suchcells include but are not limited to skin fibroblast cells, follicularcells, skeletal muscle cells, hepatocytes, and neural tissues. Suchcells also include a variety of tissue stem cells such as mesenchymalstem cells, neural stem cells, muscle stem cells, skin stem cells, andintestinal stem cells.

Example 9: Semi In Vivo Therapeutic Use of Temperature-Sensitive Agents

This example describes a semi in vivo method for transiently deliveringan RNA or protein to cells (FIG. 16). A temperature-sensitivetherapeutic agent can be any of the temperature-sensitive therapeuticagents disclosed herein. A ts-agent is functional at a permissivetemperature (e.g., 33° C.), but non-functional at a non-permissivetemperature (e.g., 37° C.).

A patient undergoes a procedure for therapeutic hypothermia: thepatient's core body temperature is maintained at a temperature lowerthan normal body temperature (e.g., 33° C.). Target cells (anycells—autologous or allogenic) are treated by the ts-agent ex vivo andimmediately infused into the patient's circulation or injected into thepatient's organs.

While the patient is maintained at the targeted temperature, e.g., 33°C. for some time, e.g., 24 hours, the ts-agent exhibits their expectedfunctions. Levels of RNAs (or proteins translated from the RNAs) of aGOI increase at a permissive temperature reach a high level.Subsequently, the patient's body temperature is returned to normaltemperature at 37° C. The ts-agent no longer functions at thenon-permissive condition, 37° C. inside the patient's body. The bodytemperature is maintained at or above 37° C. for the lifetime of thepatient, and thus, the ts-agent is not reactivated and cells other thanthe target cells will not be treated with the ts-agent. Notably, thistherapeutic procedure can be applied to any cell type including thosedescribed above.

Example 10: In Vivo Therapeutic Use of Temperature-Sensitive Agents

This example demonstrates how a temperature-sensitive viral vector isadministered to a subject and transiently activated when mildhypothermia is induced in the subject (FIG. 17). A temperature-sensitivetherapeutic agent can be any of the temperature-sensitive therapeuticagents disclosed herein. Temperature-sensitive therapeutic agents arefunctional at a permissive temperature (e.g., 33° C.), butnon-functional at a non-permissive temperature (e.g., 37° C.: a humanbody temperature).

The subject's body temperature is lowered using a target-temperaturemanagement (TTM) procedure, which has been used in the clinic forpatients with heart and brain trauma (Callaway et al., 2015). A TTMprocedure is designed to achieve and maintain a specific bodytemperature in a subject for a duration of time. Such procedures havepreviously been used therapeutically to reduce the negative effectsresulting from various acute health issues such as heart attacks andstrokes. Equipment and general methods of using a TTM procedure areknown in the art and can be used with the methods described herein. TheTTM procedure can be carried out using a number of methods, includingcooling catheters, cooling blankets, and application of ice around thebody. A variety of instruments have been used for such purposes. Forexample, the ArcticSun™ is an instrument that can be used to decrease orincrease a patient's body temperature to between 32° C.-38.5° C. (Pittlet al., 2013). The procedure can be performed safely and it has beenreported that there are no major adverse effects that are caused by thisinstrument.

A patient is placed under hypothermic conditions using the TTMprocedure, and the target body temperature is one sufficient to inducean activity of the temperature-sensitive therapeutic agent. Thetemperature-sensitive therapeutic agent is delivered directly to thepatient through either the systemic route (e.g., intravenously) orthrough direct injection into organs/tissues (e.g., catheter, orpercutaneous needle injection) (FIG. 17).

The patient's temperature is kept at the permissive temperature for atime sufficient to allow induction of a desired activity of thetemperature-sensitive therapeutic agent. The desired activity of thetemperature-sensitive agent leads to a therapeutic effect in the cellscontaining or exposed to the temperature-sensitive therapeutic agent.

After the desired therapeutic effect is achieved, the patient's bodytemperature is then returned to a normal body temperature (i.e., anon-permissive temperature) causing the activity of thetemperature-sensitive therapeutic agent to cease. This is followed bydegradation of temperature-sensitive therapeutic agent.

Systemic Delivery Through Circulation

A patient is placed under hypothermic conditions (e.g., at 33° C.). Oncethe patient's core body temperature is maintained at the targettemperature stably, a ts-agent is delivered directly to the patientintravenously. The ts-agent is delivered to many organs and tissuesthrough this systemic route. The core body temperature of the patient ismaintained at the functional temperature for a desired duration (e.g.,24 hours). While the patient's body temperature is kept at a permissivetemperature for the agent (e.g., at 33° C.), the agent is functioning.When the patient's body temperature is returned to normal at 37° C.,which is a non-permissive temperature of the agent, the agent stopsworking.

The ts-agent may be a naked RNA (i.e., a synthetic RNA). Systemicdelivery through circulation delivers a naked RNA to many organs with orwithout the target organ specificity. Alternatively, the ts-agent is anRNA (i.e., a synthetic RNA) encapsulated by nanoparticles, which areengineered to target specific cell types, tissues, organs, cancers,tumors, or abnormal cells. Thus, systemic delivery through circulationdelivers a nanoparticle-encapsulated RNA to specific cell types,tissues, organs, cancers, tumors, or abnormal cells. Alternatively, thets-agent is an RNA packaged into a viral particle. Depending on theenvelope types and other features, a virus particle targets specificcell types, tissues, organs, cancers, tumors, or abnormal cells. Thus,systemic delivery through circulation delivers an RNA packaged into aviral particle to specific cell types, tissues, organs, cancers, tumors,or abnormal cells. Alternatively, ts-agent is a temperature-sensitivevirus vector. Depending on the envelope types and other features, avirus particle targets specific cell types, tissues, organs, cancers,tumors, or abnormal cells. Thus, systemic delivery through circulationdelivers a temperature-sensitive virus vector to specific cell types,tissues, organs, cancers, tumors, or abnormal cells.

Targeting Delivery to the Brain and Spinal Cord Through CerebrospinalFluid

A patient is placed under hypothermic conditions (e.g., at 33° C.). Oncethe patient's core body temperature is maintained at the targettemperature stably, a ts-agent is delivered directly to the patient'scerebrospinal fluids by an epidural injection. The ts-agent is deliveredto the brain and spinal cord. The core body temperature of the patientcontinues to be maintained at the permissive temperature for the desiredduration (e.g., 24 hours). While the patient's body temperature is keptat a permissive temperature for the agent (e.g., at 33° C.), the agentis functioning. When the patient's body temperature is returned tonormal at 37° C., which is a non-permissive temperature of the agent,the agent stops working.

Targeting Delivery to Liver, Kidney, Skeletal Muscles, Cardiac Muscles,Pancreas, Bone Marrow, and Other Organs Through Percutaneous Injection

A patient is placed under hypothermic conditions (e.g., at 33° C.). Oncethe patient's core body temperature is maintained at the targettemperature stably, a ts-agent is injected through the skin(percutaneously) into organs such as the liver, kidney, skeletalmuscles, cardiac muscles, pancreas, or other organs using a very thinneedle with the visual guidance of ultrasound or CT. The core bodytemperature of the patient is maintained at the permissive temperaturefor the desired duration (e.g., 24 hours). While the patient's bodytemperature is kept at the permissive temperature for the agent (e.g.,at 33° C.), the agent is functioning. When the patient's bodytemperature is returned to normal at 37° C., which is a non-permissivetemperature of the agent, the agent stops working.

Targeting Delivery to Liver, Kidney, Skeletal Muscles, Cardiac Muscles,Pancreas, Bone Marrow, and Other Organs Through Endoscopy with InjectionNeedle Catheter

A patient is placed under hypothermic conditions (e.g., at 33° C.). Oncethe patient's core body temperature is maintained at the targettemperature stably, then a ts-agent is delivered directly to specificorgans and tissues through endoscopic injection needle catheter. Thecore body temperature of the patient is maintained at the permissivetemperature for the desired duration (e.g., 24 hours). While thepatient's body temperature is kept at a permissive temperature for theagent (e.g., at 33° C.), the agent is functioning. When the patient'sbody temperature is returned to normal at 37° C., which is anon-permissive temperature of the agent, the agent stops working.

Targeting Delivery to Liver, Kidney, Skeletal Muscles, Cardiac Muscles,Pancreas, Bone Marrow, and Other Organs Through Angiocatheter

A patient is placed under hypothermic conditions (e.g., at 33° C.). Oncethe patient's core body temperature is maintained at the targettemperature stably, then a ts-agent is delivered directly to specificorgans and tissues through angiocatheter. The core body temperature ofthe patient is maintained at a permissive temperature for the desiredduration (e.g., 24 hours). While the patient's body temperature is keptat a permissive temperature for the agent (e.g., at 33° C.), the agentis functioning. When the patient's body temperature is returned tonormal at 37° C., which is a non-functional temperature of the agent,the agent stops working.

Targeting Delivery to Lung and Other Organs Through Inhalation

A patient is placed under hypothermic conditions (e.g., at 33° C.). Oncethe patient's core body temperature is maintained at the targettemperature stably, then a ts-agent is delivered directly to the patientby inhalation. The ts-agent is delivered to lungs and other organsthrough via inhalation through the lungs. The core body temperature ofthe patient is maintained at a permissive temperature for the desiredduration (e.g., 24 hours). While the patient's temperature is kept at apermissive temperature for the agent (e.g., at 33° C.), the agent isfunctioning. When the patient's body temperature is returned to normalat 37° C., which is a non-permissive temperature of the agent, the agentstops working.

Targeting Delivery to Bone Marrow Cells Mobilized to Spleen

A patient will receive an injection of G-CSF, plerixafor or othercytokines to mobilize bone marrow cells (including, without limitation,CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, andendothelial stem cells) to the spleen of the subject. The patient isplaced under hypothermic conditions (e.g., at 33° C.). Once thepatient's core body temperature is maintained at the target temperaturestably, then a ts-agent is delivered to the spleen via the methodsdescribed above. Subsequently, the ts-agent is delivered to bone marrowcells mobilized to the spleen. The core body temperature of the patientis maintained at a permissive temperature for the desired duration(e.g., 24 hours). While the patient's temperature is kept at apermissive temperature for the agent (e.g., at 33° C.), the agent isfunctioning. When the patient's body temperature is returned to normaltemperature at 37° C., which is a non-permissive temperature for theagent, the agent stops working. For instance, the methods may includeadministering a therapeutically effective amount of atemperature-sensitive agent (e.g., a temperature-sensitive therapeuticagent) to one or more bone marrow cells (including, without limitation,CD34+ cells, hematopoietic stem cells, mesenchymal stem cells,endothelial stem cells) in the spleen.

Example 11: Differentiation of Human ES and iPS Cells into Desired CellTypes by Self-Replicating RNAs as Temperature-Sensitive Agents

This example describes the finding that a temperature-sensitiveself-replicating RNA expressing human transcription factors candifferentiate hPSCs into a variety of differentiated cells. The findingpresented in this example may be applied to the “ex vivo treatment ofcells with ts-agents.” Differentiated cells generated by this methodhave many useful applications, such as in vitro modeling of humandiseases by using patients-derived iPS cells and drug screening. Thefindings presented in this example may be applied to “ex vivotherapeutic use of ts-agents.” Differentiated cells generated in thismethod can be transplanted to repair defective organs and tissues inpatients. For example, neurons (more specifically dopaminergic neurons)generated ex vivo by this method are transplanted to patient'ssubstantia nigra (a part of brain) to treat Parkinson's disease. Thefindings presented in this example may be applied to “semi in vivotherapeutic use of ts-agents.” Cells treated with ts-agents that induceneurons are transplanted into a patient who undergoes therapeutichypothermia so that the ts-induced cell differentiation occurs inpatient's body. Once the differentiated cells appear, ts-agents areturned off by returning patient's body temperature to non-permissivetemperature (i.e., 37°). The findings presented in this example may beapplied to “in vivo therapeutic use of ts-agents.” For example,ts-agents expressing a set of transcription factor are directly injectedinto a pancreas of Diabetes patient who undergoes therapeutichypothermia (at a permissive temperature) so that the patient'spancreatic duct cells are converted to insulin-secreting beta-cells invivo. Once the desired cells appear, ts-agents are turned off byreturning patient's body temperature to non-permissive temperature(i.e., 37°).

Materials and Methods

Cell Culture

A human adipose stem cell-derived iPS cell line (ADSC-iPS cells) waspurchased from System Biosciences (Palo Alto, Calif.). Cells wereroutinely maintained as undifferentiated pluripotent cells according tothe standard hPSC culture method. Briefly, cells were cultured inStemFit basic02 (Ajinomoto, Japan) supplemented with 100 ng/ml FGF2.Further, cells were cultured on cell culture dishes coated with alaminin-511 substrate (iMatrix-511, Nippi, Japan).

Temperature-Sensitive Self-Replicating RNAs (srRNA1ts2)

An open reading frame of a gene of interest (GOI) was cloned intosrRNA1ts2 vector so that the expression of GOI is controlled in atemperature sensitive manner. Synthetic RNAs were produced by in vitrotranscription from the vectors according to Yoshioka et al., 2013 andused for transfection. The following GOI was cloned into the srRNA1ts2vectors:

srRNA1ts2-NGN3: human neurogenin 3 (NGN3) [NCBI GeneID: 50674]; and

srRNA1ts2-ETV2: human ETS Variant 2 (ETV2) [NCBI GeneID: 2116].

Results

Generation of Neurons

ADSC-iPSC cells were plated on a 24-well plate at the density of1.2×10{circumflex over ( )}5 cells/well. Before plating the cells, cellswere pretreated with Rock inhibitor for 1 hour. 24 hours after theplating, cells were transfected with srRNA 1ts2-NGN3. For transfection,each well of a 24-well plate was treated with, 0.5 μg synthetic RNA(srRNA) mixed with 1 μl of JetMessenger (Polyplus) transfection reagentat a final volume of 50 μl. After adding the transfection complex to thecells, 450 μl of culture media was added. The cells were incubated at33° C. for 72 hours. The cells were then passaged and cultured onornithine/laminin-coated glass coverslips. Cells were then cultured at37° C. The medium was changed every day. The srRNA1ts2-NGN3 vectorcontains a puromycin N-acetyltransferase (pac) selection gene insertedafter the “IRES” sequence, and thus, can be selected using puromycin.After passaging, cells were cultured in the presence of 1 μg/ml ofpuromycin for 24 hours. The phase-contrast images were taken on day 0,1, 2, 3, 4, 5 and 6 (FIG. 18). To show the formed neurites more clearly,a magnified picture of the day 6 image is also shown. On day 9, cellswere fixed and stained with an antibody against tubulin beta III(TUBB3)—a neural marker. Fluorescence microscopy images of two differentmagnifications (10×, 40×) are shown (FIG. 18). The results show that thesrRNA1ts2-NGN3 can differentiate human iPS cells into neurons rapidlyand efficiently.

Generation of Vascular Endothelial Cells

ADSC-iPSC cells were plated on a 24-well plate at the density of1.2×10≡cells/well. Before plating the cells, cells were pretreated withRock inhibitor for 1 hour. 24 hours after the plating, cells weretransfected with srRNA1ts2-ETV2. For transfection, each well of a24-well plate was treated with, 0.5 μg synthetic RNA (srRNA) mixed with1 μl of JetMessenger (Polyplus) transfection reagent at a final volumeof 50 μl. After adding the transfection complex to the cells, 450 μl ofculture media was added. The cells were incubated at 32° C. for 3 days,and then further cultured at 37° C. for 5 more days (8 days total). Themedium was changed every day. The srRNA1ts2-ETV2 vector contains apuromycin N-acetyltransferase (pac) selection gene inserted after the“IRES” sequence, and thus, can be selected using puromycin. 1 μg/ml ofpuromycin was added to the culture at the time of temperature switchfrom 33° C. to 37° C. The next day, the medium was replaced with mediacontaining 1 μg/ml of puromycin. Therefore, cells were cultured for 2days in the presence of 1 μg/ml of puromycin. The phase-contrast imageswere taken on day 1, 2, 3, 4, 5, 6, 7 and 8 (FIG. 19). On day 8, cellswere fixed and stained with an antibody against CD31 (a marker forvascular endothelial cells). Fluorescence microscopy images of twodifferent magnifications (10×, 20×) are shown (FIG. 19). The resultsshow that the srRNA1ts2-ETV2 can differentiate human iPS cells intovascular endothelial cells rapidly and efficiently.

Example 12: Genome Editing

Genome editing is a genetic engineering method to alter an organism'sgenome by replacing, deleting, or adding nucleotide sequences. It hasbeen proposed that genome editing can be used for correcting genomemutations for gene therapy. As a first step, genomic DNA must be cleavedat specific locations by DNA nucleases such as zinc finger nucleases(ZFNs), transcription activator-like effector-based nucleases (TALENs),or the clustered regularly interspaced short palindromic repeat(CRISPR)-CAS9 system. The introduction of these nucleases must becarefully controlled, because the double-stranded DNA breaks introducedby these enzymes are highly deleterious to cells. The targetedexpression of these nucleases requires precise control of timing andduration. It has been reported that both guide RNA and CAS9 can beencoded on a single Sendai virus vector, which makes it possible todeliver these components for genome editing of human cells at a highefficiency (Park et al., Molecular Therapy 2016). However, continuousexpression of CAS9 could cause introduction of uncontrollable DNA breaksand mutations in human cells. Thus, to use the gene editing systemtherapeutically, it is desirable to have CAS9 expressed for a shorttime, on the order of hours, not days. To this end,temperature-sensitive agents can be used as a delivery vehicle of thesecomponents, especially the nucleases.

First, the Sendai Virus Vector used for the reported CRISPR/CAS9 system(Park et al., 2016) is replaced by a temperature-sensitive Sendai ViralVector (SeVts-CAS9-guideRNA). In one embodiment, an SeVts is anSeV18/TS15ΔF (Ban et al., PNAS 2011). Human primary fibroblast cells areinfected with the temperature-sensitive Sendai Viral Vector at MOI 25 at33° C., and are maintained at 33° C. in a CO₂ incubator for 24-48 hours.Subsequently, the temperature of the cell culture is shifted to 37° C.for the rest of the cell culture. Viral replication and increased CAS9expression only occur when the cell culture is maintained at 33° C., andthus, the exposure of the cells to CAS9 nuclease is limited to thisshort time. While the cells are cultured at 37° C., thetemperature-sensitive Sendai Viral Vector is eventually lost from thecells, and thus, there will be no concern about the reactivation of CAS9expression in vitro and in vivo. Similarly, a temperature-sensitiveself-replicating RNA such as srRNA1ts2 (srRNA1ts2-CAS9-guideRNA) couldbe used in lieu of the SeVts-CAS9-guideRNA.

Example 13: CAR T Cell Therapy

CAR T cell therapy uses a transfer of vector encoding chimeric antigenreceptor (CARs) into a patient's own T cells and stably express CARs incytotoxic T cells (Maus and June 2016). CAR T cell therapy aims toreprogram the patient's own T cells to attack malignant cells in thepatient. For example, CAR T cells targeting CD19 have been appliedsuccessfully to B-cell malignancies. However, CD19 is also expressed ina normal B cells, and thus, the continuous expression of CARs couldincur the side effects, and is not desirable. Therefore, recent clinicaltrials transfer synthetic mRNAs encoding CARs into a patient's own Tcells so that the expression of CARs is transient (ClinicalTrials.govIdentifier: NCT02624258). However, a short turnover time (<12 hours) anda relatively low protein expression levels by synthetic mRNAs present aproblem in achieving sufficient expression levels of CARs. To addressthis issue, ts-agents are used as a delivery vehicle of CARs to T cells.

Ex Vivo Therapeutic Use of Temperature-Sensitive Agents to Deliver CARsto T Cells

T cells are collected from peripheral blood using an apheresis machine(COBE Spectra) and a magnetic bead-based enrichment method (Miltenyi'sCliniMacs Prodigy). Then, T cells are transfected with srRNA1ts2-CARsand cultured ex vivo at a permissive temperature (33° C.) for 24 hours(or longer). Ideally, this procedure is performed in a functionallyclosed system such as Miltenyi's CliniMacs Prodigy. Subsequently, thetreated cells are infused back into the patient. In the patient's bodyat a non-permissive temperature (37° C.), srRNA1ts2-CARs stop producingCARs, but sufficient quantities of CARs are already present on thesurface of T cells, which exert the expected functions of CARs.Alternatively, a temperature-sensitive Sendai Virus Vector encoding CARs(SeVts-CARs) is used in lieu of the srRNA1ts2-CAR. For instance, theSeVts may be a SeV18/TS15ΔF (Ban et al., PNAS 2011).

Semi In Vivo Therapeutic Use of Temperature-Sensitive Agents to DeliverCARs to T Cells

T cells treated with srRNA1ts2-CARs are immediately infused into apatient who is maintained at a permissive temperature (e.g., 33° C.) bytherapeutic hypothermia. While maintained a permissive temperature,srRNA1ts2-CARs is functional. However, when the temperature of thepatient is switched to a normal temperature (37° C.), srRNA1ts2-CARsstop producing CARs. Alternatively, a temperature-sensitive Sendai ViralVector encoding CARS (SeVts-CARs) could be used. For instance, the SeVtsmay be a SeV18/TS15ΔF (Ban et al., PNAS 2011).

In Vivo Therapeutic Use of Temperature-Sensitive Agents to Deliver CARsto T Cells

srRNA1ts2-CARs are targeted directly to T cells through in vivo deliveryto a patient's body maintained at a permissive temperature (33° C.).While maintained at a permissive temperature, srRNA1ts2-CARs isfunctional. However, when the temperature of the patient is switched toa normal temperature (37° C.), srRNA1ts2-CARs stop producing CARs.Alternatively, a temperature-sensitive Sendai Viral Vector encoding CARS(SeVts-CARs) could be used. For instance, the SeVts may be anSeV18/TS15ΔF (Ban et al., PNAS 2011).

Example 14: Dominant-Negative Mutants of PD1 and CTLA4

Programmed death-1 (PD1) and cytotoxic T-lymphocyte antigen-4 (CTLA4)are known to function as immune checkpoints. Systemic delivery ofantibodies (e.g., nivolumab and pembrolizumab) against PD1 and CTLA4 hasbeen used for cancer therapy. However, up to 20% of patients who receivethese therapies experienced adverse events, such as autoimmunity(Roberts et al., 2017). Because the immune checkpoints naturallyfunction to restrict the activation of immune functions to preventautoimmunity, systemically blocking the function of these molecules bythe administration of antibodies is considered to activate immunityagainst not only cancers, but also patient's own normal cells. Toaddress this issue, dominant negative mutants of PD1 and CTLA1 areexpressed specifically in T cells, and thereby block the function of PD1and CTLA4 only in T cells (Shin et al., 2016). For PD1, it has beenshown that a mutant that contains an extracellular domain andtransmembrane domain, but lacks cytoplasmic domain, functions as adominant negative mutant (PD1 decoy or PD1Δ) (Shin et al., 2016).Although a retrovirus vector was used to deliver the PD1 decoy toisolated T cells in a mouse study, the integration of a retrovirusvector into the host genome and the persistent expression of the PD1decoy is not desirable for humans, considering potential adverse effectsof long-term blocking the immune checkpoints. Ideally, the expression ofa PD1 decoy in T cells (or other immune cells) should be permanentlyturned off after exerting its beneficial functions. To this end,ts-agents are used as a delivery vehicle of the dominant-negativemutants of PD1 and CTLA4. Ex Vivo Therapeutic Use ofTemperature-Sensitive Agents to Deliver a Dominant-Negative PD1 Mutant

Target cells (e.g., T cells) are collected from peripheral blood usingan apheresis machine (COBE Spectra) and a magnetic bead-based enrichmentmethod (Miltenyi's CliniMacs Prodigy). Then, the target cells aretransfected with srRNA1ts2-PD1Δ and cultured at a permissive temperature(e.g., 33° C.) for a desired amount of time (e.g., 24 hours or 1 week).Subsequently, the treated cells are infused into patients. In thepatient's body at non-permissive temperature (37° C.), srRNA1ts2-PD1Δwill eventually stop producing PD1Δ. However, as long as the PD1Δprotein exists in the target cells, PD1 functions are blocked.Alternatively, a temperature-sensitive Sendai Virus Vector encoding PD1Δcould be used in lieu of srRNA1ts2-PD1Δ. For instance, the SeVts may bean SeV18/TS15ΔF (Ban et al., PNAS 2011).

Semi In Vivo Therapeutic Use of Temperature-Sensitive Agents to Delivera Dominant-Negative PD1 Mutant

T cells treated with srRNA1ts2-PD1Δ are immediately infused into apatient who is maintained at a permissive temperature (e.g., 33° C.) bytherapeutic hypothermia. While maintained at a permissive temperature,srRNA1ts2-PD1Δ is functional. However, when the temperature of thepatient is switched to a normal temperature (37° C.), srRNA1ts2-PD1Δstop producing PD1Δ. Alternatively, a temperature-sensitive Sendai VirusVector encoding PD1Δ could be used in lieu of srRNA1ts2-PD1Δ. Forinstance, the SeVts may be an SeV18/TS15ΔF (Ban et al., PNAS 2011).

In Vivo Therapeutic Use of Temperature-Sensitive Agents to Deliver aDominant-Negative PD1 Mutant

srRNA1ts2-PD1Δ are targeted directly to T cells through in vivo deliveryinto a patient's body maintained at a permissive temperature (33° C.).While maintaining a permissive temperature, srRNA1ts2-PD1Δ isfunctional. However, when the temperature of the patient is switched toa normal temperature (37° C.), srRNA1ts2-PD1Δ stop producing PD1Δ.Alternatively, a temperature-sensitive Sendai Virus Vector encoding PD1Δcould be used in lieu of srRNA1ts2-PD1Δ. For instance, the SeVts may bean SeV18/TS15ΔF (Ban et al., PNAS 2011).

Example 15: Combination Therapy of CAR T Cell and Dominant-NegativeMutants of PD1 and CTLA4

Systemic administration of PD1-blocking antibodies enhances theeradication of tumors by CAR T cells (John et al., 2013). Ts-agents,particularly srRNA1ts2, provide a delivery vehicle for both PD1-blockingfunction and CARs to the same T cells, because srRNA1ts2 has a largecargo capacity and can accommodate multiple genes in the same vector. ExVivo Therapeutic Use of Temperature-Sensitive Agents to Deliver CARs andDominant-Negative PD1 to T Cells

The coding regions of PD1Δ and CARs are fused with the insertion of P2Apeptide (self-cleaving peptide) between them. This fusion protein-codingsequence is now inserted in srRNA1ts2 vector as GOI. Synthetic RNAs(srRNA1ts2-PD1Δ-CARs) are generated from the srRNA1ts2-PD1Δ-CARs vector.T cells are collected from peripheral blood using an apheresis machine(COBE Spectra) and a magnetic bead-based enrichment method (Miltenyi'sCliniMacs Prodigy). Then, T cells are transfected withsrRNA1ts2-PD1Δ-CARs and cultured at a permissive temperature (33° C.)for 24 hours (or longer). Subsequently, the treated cells are infusedback into the patient. In the patient's body, at a non-permissivetemperature (37° C.), srRNA1ts2-PD1Δ-CARs stop producing PD1Δ and CARs.However, a sufficient quantity of PD1Δ and CARs are already present onthe surface of the T cells. Alternatively, a temperature-sensitiveSendai Virus Vector encoding PD1Δ-CARs can be used in lieu ofsrRNA1ts2-PD1Δ-CARs. For instance, the SeVts may be an SeV18/TS15ΔF (Banet al., PNAS 2011).

Semi In Vivo Therapeutic Use of Temperature-Sensitive Agents to DeliverCARs and Dominant-Negative PD1 to T Cells

T cells treated with srRNA1ts2-PD1Δ-CARs are immediately infused into apatient maintained at permissive temperature (e.g., 33° C.) bytherapeutic hypothermia. While maintained at a permissive temperature,srRNA1ts2-PD1Δ-CARs is functional. However, when the temperature of thepatient is switched to a normal temperature (37° C.),srRNA1ts2-PD1Δ-CARs stop producing CARs and PD1Δ. Alternatively, atemperature-sensitive Sendai Virus Vector encoding PD1Δ-CARs can be usedin lieu of srRNA1ts2-PD1Δ-CARs. For instance, the SeVts may be anSeV18/TS15ΔF (Ban et al., PNAS 2011).

In Vivo Therapeutic Use of Temperature-Sensitive Agents to Deliver CARsand Dominant-Negative PD1 to T Cells

srRNA1ts2-PD1Δ-CARs are targeted directly to T cells through in vivodelivery into a patient's body maintained at a permissive temperature(33° C.). While maintained at a permissive temperature,srRNA1ts2-PD1Δ-CARs is functional. However, when the temperature of thepatient is switched to a normal temperature (37° C.),srRNA1ts2-PD1Δ-CARs stop producing CARs and PD1Δ. Alternatively, atemperature-sensitive Sendai Virus Vector encoding PD1Δ-CARs can be usedin lieu of srRNA1ts2-PD1Δ-CARs. For instance, the SeVts may be anSeV18/TS15ΔF (Ban et al., PNAS 2011).

Example 16: Ribonucleoproteins

Ribonucleoproteins function by forming a complex with RNA. Therapeuticapplication of ribonucleoproteins is challenging because it is difficultto express both a protein and RNA from the same vector. For example,major components of telomerase are human telomerase reversetranscriptase (TERT) and telomerase RNA (TERC). Abnormal shortening oftelomeres causes diseases. Thus, delivery of telomerase (TERT+TERC) toextend telomeres is a desirable therapeutic intervention. However, thepersistent presence of telomerase could cause adverse events such astumor formation. Therefore, it is desirable to have a time-limiteddelivery of both TERT and TERC. To this end, ts-agents can be used as adelivery vehicle of TERT and TERC.

Ex Vivo Therapeutic Use of Temperature-Sensitive Agents to Deliver TERTand TERC

srRNA1ts2 vector is constructed to express a protein TERT in atemperature-sensitive manner. The vector also contains TERC, RNAcomponent sandwiched by self-cleaving ribozymes (e.g., Hammerheadribozyme). The resulting vector is used to generate synthetic RNA(srRNA1ts2-TERT-TERC). Target cells (e.g., hematopoietic stem cells) aretransfected with srRNA1ts2-TERT-TERC. At a permissive temperature (e.g.,33° C.), srRNA1ts2-TERT-TERC replicates and produces TERT (proteins). Atthe same time, some of the RNA molecules (srRNA1ts2-TERT-TERC) isself-cleaved by ribozymes and becomes TERC (RNAs). Then, the TERT andTERC form a telomerase complex and extend telomeres. The target cells(e.g., hematopoietic stem cells) are then infused into a patient'scirculation. At 37° C., a non-permissive temperature,srRNA1ts2-TERT-TERC stops working. Alternatively, atemperature-sensitive Sendai Virus Vector encoding TERT-TERC can be usedin lieu of srRNA1ts2-TERT-TERC. For instance, the SeVts may be anSeV18/TS15ΔF (Ban et al., PNAS 2011).

Semi In Vivo Therapeutic Use of Temperature-Sensitive Agents to DeliverTERT and TERC

Target cells treated with srRNA1ts2-TERT-TERC are immediatelytransplanted to a patient maintained at a permissive temperature (e.g.,33° C.) by therapeutic hypothermia. While maintained at a permissivetemperature, srRNA1ts2-TERT-TERC is functional. However, when thetemperature of the patient is switched to a normal temperature (37° C.),srRNA1ts2-TERT-TERC stops producing TERT and TERC. Alternatively, atemperature-sensitive Sendai Virus Vector encoding TERT-TERC can be usedin lieu of srRNA1ts2-TERT-TERC. For instance, the SeVts may be anSeV18/TS15ΔF (Ban et al., PNAS 2011).

In Vivo Therapeutic Use of Temperature-Sensitive Agents to Deliver TERTand TERC

srRNA1ts2-TERT-TERC is targeted directly to cells through in vivodelivery into a patient's body maintained at a permissive temperature(33° C.). While maintained at a permissive temperature,srRNA1ts2-TERT-TERC is functional. However, when the temperature of thepatient is switched to a normal temperature (37° C.),srRNA1ts2-TERT-TERC stops producing TERT and TERC. Alternatively, atemperature-sensitive Sendai Virus Vector encoding TERT-TERC can be usedin lieu of srRNA1ts2-TERT-TERC. For instance, the SeVts may be anSeV18/TS15ΔF (Ban et al., PNAS 2011).

Example 17: Gene Knockdown or Silencing

RNAi, including microRNA, siRNA, and shRNA has become an attractivechoice for knocking down the function of specific genes for therapeuticpurposes (Kaczmarek et al., 2017). However, a drawback of RNAitechnology is its very short-time action, which requires repeated dosingof a large quantity of siRNAs. On the other hand, although plasmids andviruses can provide a strong expression of shRNA from PolIII promoter,it is difficult to stop expression when required. Further, a DNA-basedshRNA expression system may be integrated into an organisms' genome andcause mutations. In this sense, a temperature-sensitive srRNA or Sendaivirus vector may provide an ideal solution, as they providefootprint-free (no integration into an organism's genome) controllableand prolonged expression of RNAi.

shRNA. An shRNA is incorporated in srRNAts or SeVts as a GOI flanked bya self-cleaving ribozyme, in a manner similar to a guide RNA flanked bya self-cleaving ribozyme shown in Park et al., 2016. An srRNA1ts2-shRNAis delivered to target cells, where a target gene is silenced when thetarget cells are maintained at a permissive temperature. However, whenthe temperature is shifted to non-permissive temperature, the productionof shRNA stops, and the target gene is unsilenced. Alternatively, atemperature-sensitive Sendai Virus Vector encoding shRNA can be used inlieu of srRNA1ts2-shRNA. For instance, the SeVts may be an SeV18/TS15ΔF(Ban et al., PNAS 2011).

dsRNA. An expression unit of a long double-stranded RNA (dsRNA) is madeby connecting a long sense-strand and its antisense-strand of a targetRNA (i.e., to be knocked down) by a short linker RNA sequence. Thisexpression unit of dsRNA is inserted in a srRNA1ts2 vector as a GOIflanked by a self-cleaving ribozyme, in a manner similar to a guide RNAflanked by a self-cleaving ribozyme (Park et al., 2016 and Shinagawa T1,Ishii S. 2003). To facilitate the formation of siRNAs from the dsRNA,the srRNA1ts2 vector also expresses human DICER1 (NCBI ReferenceSequence: NG_016311.1). An srRNA1ts2-dsRNA or srRNA1ts2-DICER1-dsRNA isdelivered to target cells, where a target gene is silenced when thetarget cells are maintained at a permissive temperature. However, whenthe temperature is shifted to non-permissive temperature, the productionof dsRNA (and DICER1, if present) stops, and the target gene isunsilenced. Alternatively, a temperature-sensitive Sendai Virus Vectorencoding dsRNA or dsRNA-DICER1 can be used in lieu of srRNA1ts2-dsRNA orsrRNA1ts2-dsRNA-DICER1. For instance, the SeVts may be an SeV18/TS15ΔF(Ban et al., PNAS 2011).

asRNA. An antisense-strand of a target RNA (asRNA) (i.e., to be knockeddown) is inserted in a srRNA1ts2 vector as a GOI flanked by aself-cleaving ribozyme, in a manner similar to a guide RNA flanked by aself-cleaving ribozyme shown in Park et al., 2016 and Shinagawa T1,Ishii S. 2003. To facilitate the formation of siRNAs from the dsRNA,srRNA1ts2 vector also expresses human DICER1 (NCBI Reference Sequence:NG_016311.1). An srRNA1ts2-asRNA or srRNA1ts2-DICER1-asRNA is deliveredto target cells, where a target gene is silenced when the target cellsare maintained at a permissive temperature. However, when thetemperature is shifted to non-permissive temperature, the production ofa sRNA (and DICER1, if present) stops, and the target gene isunsilenced. Alternatively, a temperature-sensitive Sendai Virus Vectorencoding asRNA or asRNA-DICER1 can be used in lieu of srRNA1ts2-asRNA orsrRNA1ts2-asRNA-DICER1. For instance, the SeVts may be an SeV18/TS15ΔF(Ban et al., PNAS 2011).

The gene knockdown or gene silencing method detailed above is applied toany of the “ex vivo treatment of cells with ts-agents,” “ex vivotherapeutic use of ts-agents”, “semi in vivo therapeutic use ofts-agents”, or “in vivo therapeutic use of ts-agents.”

Example 18: Cell Fusion Therapy

One of the strategies in the field of regenerative medicine is todifferentiate human pluripotent stem cells such as embryonic stem (ES)cells and induced pluripotent stem (iPS) cells into desired cell typessuch as neurons and muscles, and then transplant these differentiatedcells into patients. Often, it is desirable to use iPS cells that aremade from the patient's own cells such as blood cells or fibroblastcells. For example, to treat a patient with muscular dystrophy, skeletalmuscle cells that are differentiated ex vivo from ES cells or iPS cellscan be transplanted to patient's skeletal muscles. One of the technicalchallenges is to ensure the proper engraftment of exogenous muscle cellsand the replacement or supplementation of patient's defective musclefunctions. To address this issue, RNAs that encode fusogenic proteinscan be delivered into the exogenous muscle cells, which facilitates thecell-to-cell fusion among the exogenous muscle cells and a patient's ownmuscle cells.

Semi In Vivo Therapeutic Use of Temperature-Sensitive Agents to DeliverFusogenic Proteins

srRNA1ts2 vector is constructed to express fusogenic proteins such asSendai Virus F and HN proteins. F and HN are fused into a single proteinvia P2A self-cleaving peptide. The srRNA1ts2-F-HN vector is used togenerate synthetic RNA (srRNA1ts2-F-HN). It has been well establishedthat the presence of F and HN proteins on the cell surface can inducecell fusion (Rawling et al., 2008). Alternatively, human respiratorysyncytial virus F protein, which alone can induce cell-to-cell fusions(Rawling et al., 2008), is cloned into srRNA vectors to generatesynthetic RNA (srRNA1ts2-RSVF). Another example of fusogenic proteinsare Myomaker (Mymk) and Myomixer (Mymx) (also known as Myomerger) (Bi etal., 2017). Myomaker and myomixer are fused into a single protein viaP2A self-cleaving peptide. The srRNA1ts2-Mymk-Mymx vector is used togenerate synthetic RNA (srRNA1ts2-Mymk-Mymx). The presence of bothMyomaker and Myomixer induces the cell-to-cell fusion, not only muscles,but in fibroblast cells as well (Bi et al., 2017). Human iPS cells aredifferentiated into skeletal muscles using the method described above.Skeletal muscles are then transfected with srRNA1ts2-HN-F orsrRNA1ts2-SRVF or srRNA1ts2-Mymk-Mymx, then immediately injected intothe skeletal muscles of a patient, whose body temperature is alreadylowered to 33° C. by a procedure for therapeutic hypothermia (FIG. 15).While the patient is maintained at the targeted temperature (33° C.) for24 hours, the fusogenic proteins are expressed in the transplantedskeletal muscle cells, which are then fused to a patient's own defectiveskeletal muscle cells. Subsequently, the patient's body temperature isreturned to normal temperature at 37° C. The ts-agent and fusogenicproteins no longer function in this non-functional condition at 37° C.inside patient's body.

Tissues that can be treated with this semi in vivo use of ts-agents arenot limited to skeletal muscles. Cardiomyocytes and hepatocytes areusually polyploidy, and therefore, heart tissues and livers are suitabletargets for this therapy. Cardiomyocytes and hepatocytes are tissuesthat can be easily generated from human ES/iPS cells. Furthermore, semiin vivo use of ts-agents can be used to treat neurological diseases suchas spinal cord injury and neurodegenerative diseases such as Parkinson'sdiseases. Alternatively, a temperature-sensitive Sendai Virus Vector canbe used in lieu of srRNA1ts. For instance, the SeVts may be anSeV18/TS15ΔF (Ban et al., PNAS 2011).

The gene knockdown or gene silencing method detailed above may beapplied to any of the “ex vivo treatment of cells with ts-agents,” “exvivo therapeutic use of ts-agents”, “semi in vivo therapeutic use ofts-agents”, or “in vivo therapeutic use of ts-agents.”

Example 19: Vaccines

Temperature-sensitive agents (ts-agents) such as srRNAs or Sendai virusvectors, are functional at a permissive temperature (e.g., about 31-34°C.), but non-functional at a non-permissive temperature (e.g., ≥37° C.).While the core body temperature of a human subject is about 37° C., thesurface body temperature of a human subject is about 31-34° C. Thus,ts-agents administered to cells at or near the surface of a body of ahuman patient (e.g., intradermally, subcutaneously, or intramuscularly)are functional without lowering the core body temperature of the humanpatient (FIG. 20). No further action is required.

Similarly, the temperature of the nasal cavity and upper trachea of ahuman subject is about 32° C., and the temperature of the subsegmentalbronchi of a human subject is about 35° C. (McFadden et al., 1985). Assuch, ts-agents administered intranasally to cells of the upperrespiratory tract (nasal cavity, pharnyx, and/or larnyx) and/or uppertrachea of a human patient are functional without lowering the core bodytemperature of the human patient (FIG. 22). Intranasal administrationmay be done by insufflation, inhalation or instillation. No furtheraction is required.

Alternatively, the ts-agent administered to cells at or near the surfaceof a body of a human patient (e.g., intradermally, subcutaneously, orintramuscularly) can subsequently be rendered non-functional by raisingthe surface body temperature of the human patient, for instance byapplication of a heat patch or heating pad to the treated area of thepatient's skin, soaking in a warm bath, or sitting in a hot sauna. Thistherapeutic procedure is very safe in that the ts-agent is onlyfunctional in the intended area and is non-functional in other areas ofa patient's body. Similarly, the ts-agent administered intranasally tocells of the upper respiratory tract (nasal cavity, pharnyx, and/orlarnyx) and/or upper trachea of a human patient can be renderednon-functional by placing the human patient in an environment with anon-permissive temperature (e.g., ≥37° C.).

Immunogenic compositions and vaccines employing srRNA1ts2 as a vectorare suitable for inducing an immune response against all types ofpathogens. For instance, recombinant srRNA1ts2 vectors can beconstructed relatively quickly once a coding region of an antigen of apathogen is known. Additionally, RNA of a srRNA1ts2 vector can betranscribed in vitro without the use of materials of animal or humanorigin. In this way, vaccines employing srRNA1ts2 vectors are easilyadapted to production using current good manufacturing practice.Alternatively, a temperature-sensitive Sendai virus vector encoding anantigen of a pathogen can be employed (e.g., SeV18/TS15ΔF).

The construction of srRNA1ts2 is described above in Example 3. In brief,srRNA1ts2 comprises a Venezuelan equine encephalitis virus (VEEV)replicon lacking a VEEV structural protein coding region. The VEEVreplicon comprises a VEEV nonstructural protein coding region with aninsertion of 15-18 nucleotides resulting in expression of anonstructural Protein 2 (nsP2=helicase proteinase) comprising 5 or 6additional amino acids (SEQ ID NO:39=TGAAA) between beta sheet 5 andbeta sheet 6 of the nsP2. The additional amino acids result intemperature-sensitivity of the self-replicating RNA.

The genomes of exemplary srRNA1ts2 vectors encoding the spike protein(or a portion thereof) of severe acute respiratory syndromecoronavirus-2 (SARS-CoV-2, also known as 2019-nCoV) are shown in FIG.21. The spike protein and the receptor-binding domain (RBD) of the spikeprotein of a related coronavirus were previously identified as targetsfor vaccine and drug development (Du et al., Nat Rev Microbiol,7:226-236, 2009). The sequence of the RNA genome of 2019-nCoV is setforth in NCBI Accession No.: NC_045512. Three distincttemperature-sensitive srRNA1ts2 vectors were constructed.srRNA1ts2-2019-nCoV-Spike encodes the full length spike protein.srRNA1ts2-2019-nCoV-RBD1 encodes a CD5 signal peptide fused to the RBDof the spike protein. srRNA1ts2-2019-nCoV-RBD2 encodes a signal peptideof the spike protein fused to the RBD, transmembrane domain, andcytoplasmic tail of the spike protein. Expression of the spike proteinor fragment thereof is driven by the 26S promoter of the VEEV replicon.

The entire RNAs are transcribed in vitro using T7 RNA polymerase. Then,the RNAs are transfected into cells of a subject's dermal tissue. Asuitable method for transfection is by patch electroporation of nakedRNAs. Alternatively, a microneedle is used to transfect RNAsintradermally. For instance, a dissolvable microneedle made withhyaluronic acid or a chitosan-hyaluronic acid complex is used totransfect RNAs intradermally In some embodiments, a regular Mantouxprocedure can be used. Alternatively, a special injection devicedesigned to facilitate the intradermal injection can be used.

The amino acid sequence of the spike protein ofsrRNA1ts2-2019-nCoV-Spike is set forth as SEQ ID NO:41:MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT. The signal peptide extends from residues 1-15, theextracellular region extends from residues 16-1213, the transmembranedomain extends from residues 1214-1236, and the cytoplasmic domainextends from residues 1237-1273.

The amino acid sequence of the spike protein fragment ofsrRNA1ts2-2019-nCoV-RBD1 is set forth as SEQ ID NO:42:MPMGSLQPLATLYLLGMLVASCLGPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAP. The CD5 signal peptide extendsfrom residues 1-24 and the RBD extends from residues 25-192.

The amino acid sequence of the spike protein fragment ofsrRNA1ts2-2019-nCoV-RBD2 is set forth as SEQ ID NO:43:MFVFLVLLPLVSSQCPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT. The signal peptide extends from residues1-15, the RBD extends from residues 16-207, the transmembrane domainextends from residues 208-230, and the cytoplasmic domain extends fromresidues 231-267.

The amino acid sequence of the RBD is set forth as SEQ ID NO:44:

PNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAP.

Example 20: In Vivo Expression of GOI after Injection of TemperatureSensitive-srRNA

As described in Example 19 and shown in FIG. 20, temperature-sensitiveagents (ts-agents), such as srRNAs or Sendai virus vectors, which arefunctional at a permissive temperature (e.g., about 31-34° C.), butnon-functional at a non-permissive temperature (e.g., 37° C.), can bedelivered to the surface of a human body (e.g., skin) for controlledexpression of a gene of interest (GOI). Thus, ts-agents encoding a GOIhave an inherent safety feature in that expression of the GOI is limitedto the local area (permissive temperature) to which the ts-agent isdelivered. That is, unintended expression of a GOI by a ts-agent doesnot occur in areas of a subject's body that naturally have a temperatureabove or below the permissive temperature. This example demonstratesthat this safety feature works in vivo in a model mammalian subject,namely mice. The skin temperature of mice is similar to that of humans(Mortola 2013), and thus intradermal delivery of ts-agents to mice isexpected to mimic intradermal delivery of ts-agents to humans.

RNAs were formulated as naked RNAs, without lipid nanoparticles or anyother transfection reagents, in lactated Ringer's solution. RNA (5 μg)encoding luciferase (LUC) was injected intradermally into a single siteon the right hind limb of CD-1 outbred mice. Luciferase activity wasvisualized and quantitated by using a bioluminescent Imaging system, AMIHTX (Spectral Instruments Imaging, Tucson, Ariz.).

FIG. 23 shows the time-course of in vivo luciferase activity from Day 0(injection day) through Day 26 in recipients of: a control synRNA-LUCfrom TriLink (San Diego, Calif.); or a temperature-sensitive srRNA(srRNA1ts2-LUC). Luciferase imagining showed that intradermal injectionof naked RNA encoding luciferase resulted in expression of luciferase invivo. Strikingly, the in vivo expression of luciferase driven bysrRNA1ts2-LUC, due to its self-replicating feature, continued for nearlya month. In contrast, the in vivo expression of luciferase driven bysynRNA-LUC continued for only a little over a week. Furthermore, theexpression level of luciferase in recipients of srRNA1ts2-LUC, due toits self-replicating feature, was 10- to 100-fold higher than theexpression level of luciferase in recipients of synRNA-LUC. Importantly,luciferase expression was not observed in uninjected areas of therecipients' skin, or within internal organs of the recipients. Thisobservation indicates that the temperature-sensitive srRNA1ts2-LUC didnot replicate and express luciferase under non-permissive conditions.

Example 21: Cellular Immunity Elicited by Temperature-Sensitive srRNAVaccine

In this example, cytokine-secreting splenocytes elicited by intradermaladministration of temperature-sensitive srRNA expressing the receptorbinding domain (RBD) of the spike protein of SARS CoV-2 were measured.RNAs were formulated as naked RNAs, without any lipid nanoparticles orany other transfection reagents, in lactated Ringer's solution. Toassess cellular immunity, the enzyme-linked immunospot (ELISpot) assay,which quantitates the number of cytokine secreting cells, was performedon splenocytes obtained from CD-1 outbred mice that received a singledose of a placebo (buffer only) or 5 μg, 25 μg, or 100 μg ofsrRNA1ts2-2019-CoV-RBD1 RNA (described in Example 19). Splenocytesisolated 12 days post injection were stimulated for 24 hours with a poolof 53 peptides (15mers with 11 amino acid overlaps) that coverSARS-CoV-2 RBD (PepMix SARS-CoV-2 [S-RBD], JPT Peptide TechnologiesGmbH, Berlin, Germany).

As shown in FIG. 24A, srRNA1ts2-2019-nCoV-RBD1 administered byintradermal injection induced cellular immunity against SARS-CoV-2 RBDin a dose-dependent manner. IFN-γ-secreting cells (FIG. 24A), which arecharacteristic of type 1 CD4+T helper cells (Th1 cells) and CD8+cytotoxic T cells, were preferentially expanded by thetemperature-sensitive SARS-CoV-2 RBD srRNA vaccine. In contrast,IL4-secreting cells (FIG. 24B), which are characteristic of type 2 CD4+Thelper cells (Th2 cells), were not expanded by the temperature-sensitiveSARS-CoV-2 RBD srRNA vaccine. In conclusion, the results showed thatintradermal administration of srRNA1ts2-2019-nCoV-RBD1 elicited a Th1dominant (Th1>Th2) cellular immune response against SARS-CoV-2 RBD,which is a desirable feature of a vaccine directed against a viralpathogen.

Example 22: Humoral Immunity Elicited by Temperature-Sensitive srRNAVaccine

In this example, antibodies elicited by intradermal administration oftemperature-sensitive srRNA expressing the receptor binding domain (RBD)of the spike protein of SARS CoV-2 were measured. RNAs were formulatedas naked RNAs, without any lipid nanoparticles or any other transfectionreagents, in lactated Ringer's solution. Groups of CD-1 outbred mice(N=10) received one of three formulations by intradermal injection onDay 0 and Day 14 (black triangles): a placebo (buffer only) (FIG. 25A),5 μg of temperature-sensitive srRNA1ts2-2019-CoV-RBD1 RNA (FIG. 25B), or5 μg of temperature-sensitive srRNA1ts2-2019-CoV-RBD1 RNA in combinationwith a RNase inhibitor (3 units of RNasin Plus) (Promega, Madison, Wis.)(FIG. 25C). All mice received a recombinant RBD protein (Ala319-Phe541,with a C-terminal 6-His tag, Accession #YP_009724390.1: R&D Systems,Minneapolis, Minn.) by intradermal injection on Day 49 (open triangles).To assess humoral immunity, an enzyme-linked immunosorbent assay(ELISA), which quantitates the amount of immunoglobulin G (IgG) specificto a recombinant RBD protein (represented by the measurement at OD450),was performed on serum obtained from injected mice on Day −3, Day 14,Day 28, Day 46, Day 56, and Day 63. Amount of IgG in serum is shown asOD450.

As shown in FIGS. 25A-25C, an increase in RBD-specific IgG was notobserved in any group after only the prime and first booster. However,when mice received a second booster comprising a recombinant RBDprotein, the groups that received a prime and first booster comprisingsrRNA1ts2-2019-CoV-RBD1 quickly responded with an increase inRBD-specific IgG (FIGS. 25B-C), whereas the placebo group did not showan appreciably increase in RBD-specific IgG (FIG. 25A). The rapidincrease in RBD-specific IgG upon the injection of a recombinant RBDprotein indicates that the mice that received a prime and first boostercomprising srRNA1ts2-2019-CoV-RBD1, maintained immune memory, andexhibited a secondary humoral response to a recombinant RBD.

What is claimed:
 1. A composition for stimulating an immune responseagainst an antigen in a mammalian subject, comprising an excipient and atemperature-sensitive agent (ts-agent) encoding the antigen, wherein thets-agent is a temperature-sensitive Sendai viral vector, and wherein thets-agent is capable of expressing the antigen at a permissivetemperature but not at a non-permissive temperature, and the antigen isa spike protein or fragment thereof of a coronavirus.
 2. The compositionof claim 1, wherein the coronavirus is 2019-nCoV and the antigencomprises a receptor-binding domain (RBD) of the 2019-nCoV.
 3. Thecomposition of claim 1, wherein the permissive temperature is from 31°C. to 35° C., and the non-permissive temperature is 37° C.±0.5° C.
 4. Acomposition for stimulating an immune response against an antigen in amammalian subject, comprising an excipient and a temperature-sensitiveagent (ts-agent) encoding the antigen, wherein the ts-agent is atemperature-sensitive self-replicating RNA comprising an Alphavirusreplicon lacking a viral structural protein coding region, and whereinthe ts-agent is capable of expressing the antigen at a permissivetemperature but not at a non-permissive temperature, and the antigen isa spike protein or fragment thereof of a coronavirus.
 5. The compositionof claim 4, wherein the Alphavirus is selected from the group consistingof a Venezuelan equine encephalitis virus, a Sindbis virus, and aSemliki Forrest virus.
 6. The composition of claim 4, wherein theAlphavirus is a Venezuelan equine encephalitis virus.
 7. The compositionof claim 4, wherein the coronavirus is 2019-nCoV and the antigencomprises a receptor-binding domain (RBD) of the 2019-nCoV.
 8. Thecomposition of claim 4, wherein the permissive temperature is from 31°C. to 35° C., and the non-permissive temperature is 37° C.±0.5° C.
 9. Atemperature-sensitive agent (ts-agent), wherein the is-agent is atemperature-sensitive self-replicating RNA comprising an Alphavirusreplicon lacking a viral structural protein coding region, and whereinthe ts-agent comprises a nonstructural protein coding region with aninsertion of 12-18 nucleotides resulting in expression of anonstructural Protein 2 (nsP2) comprising from 4 to 6 additional aminoacids between beta sheet 5 and beta sheet 6 of the nsP2.
 10. Thetemperature-sensitive agent of claim 9, wherein the additional aminoacids comprise one sequence selected from the group consisting of SEQ IDNO:38 (GCGRT), SEQ ID NO:39 (TGAAA), and SEQ ID NO:40 (LRPHP).
 11. Thetemperature-sensitive agent of claim 9, wherein the additional aminoacids comprise the sequence of SEQ ID NO:39 (TGAAA).
 12. Thetemperature-sensitive agent of claim 9, wherein the amino acid sequenceof the nsP2 comprises one sequence selected from the group consisting ofSEQ ID NOs:29-36.
 13. The temperature-sensitive agent of claim 9,wherein the ts-agent encodes an antigen of a pathogen, and the ts-agentis capable of expressing the antigen at a permissive temperature but notat a non-permissive temperature.
 14. The temperature-sensitive agent ofclaim 13, wherein the antigen is a viral antigen and the viral antigenis not an Alphavirus antigen.
 15. The temperature-sensitive agent ofclaim 9, wherein the Alphavirus is selected from the group consisting ofa Venezuelan equine encephalitis virus, a Sindbis virus, and a SemlikiForrest virus.
 16. The temperature-sensitive agent of claim 9, whereinthe Alphavirus is a Venezuelan equine encephalitis virus.
 17. Thetemperature-sensitive agent of claim 9, wherein the ts-agent encodes apharmaceutical agent, and wherein the ts-agent is capable of expressingthe pharmaceutical agent at a permissive temperature but not at anon-permissive temperature.
 18. The temperature-sensitive agent of claim17, wherein the permissive temperature is from 31° C. to 35° C., and thenon-permissive temperature is 37° C.±0.5° C.
 19. Thetemperature-sensitive agent of claim 18, wherein the pharmaceuticalagent comprises a protein.
 20. The temperature-sensitive agent of claim18, wherein the pharmaceutical agent is selected from the groupconsisting of a non-coding RNA, a siRNA, a shRNA, and an endonucleaseediting system.